Introduction

Salinity is one of the most common environmental stress factors, which has a great impact on plant growth, metabolism, and even its distribution (Li 2002). Plant subjected to salt stress was threatened by water stress, decline of photosynthetic performance, and ion imbalance. Changes in ionic homeostasis or water stress lead to certain damages at the molecular and cellular levels of plant, further causing growth arrest. Decline in photosynthetic capacity negatively affected the accumulation of dry matter. (Guo et al. 2011; He and Zhu 2008; Tang et al. 2007). Additionally, salt stress produced plenty of reactive oxygen species (ROS) which stimulated the oxidative damage of lipids, proteins, and nucleic acids, even caused the degradation of membrane system (Demiral and Türkan 2005). For mitigating salt stress damage, salicylic acid (SA) was used in many plants (Ananieva et al. 2004; El-Tayeb 2005; Guo et al. 2011). Salicylic acid was considered to be an endogenous signal of phenolic compound. It could induce plant resistance to stress environment (Pál 2013). Existing study reported SA was helpful to induce antioxidase activity, adjust the ion absorption, promote secondary metabolites, and alleviate negative effects of salinity on the growth of different plants (Ananieva et al. 2004; Lim et al. 2012; Yildirim et al. 2008). Therefore, SA was expected to be a potential growth regulator of plant under stress.

Artemisia annua L. (Asteraceae) is an annual herb used for the treatment of fever and malaria (Baraldi et al. 2008; Ma et al. 2007). It is widely distributed in most areas of China and is mainly cropped in southwest China (Wei et al. 2005). Previous studies on the A. annua response to salinity mainly concentrated on photosynthetic performance, cell oxidation damage, and artemisinin content. Less information on ionic homeostasis and protection from salt damage was reported (Aftab et al. 2011; Prasad et al. 1998; Qureshi et al. 2005). Antioxidant response of A. annua to salt stress also remained to be elucidated.

The present study is designed to investigate the impact of salt-induced stress on A. annua growth, antioxidase activity, proline, and mineral element accumulation. Meanwhile, the influence of SA on the physiological response of A. annua grown in saline stress was evaluated in the study. All these will provide some practical basis for the cultivation of A. annua.

Materials and methods

Plant materials and stress treatments

Seeds of A. annua were sterilized with 75 % ethyl alcohol for 3 min and germinated on a filter paper in Petri dishes in a growth chamber at controlled conditions (15–25 °C temperature, 70–90 % relative humidity, 16–8 h night–day photoperiod). Seeds germinated after a week. Five seedlings each were transferred to plastic pots; each experimental pot (25-cm diameter × 25-cm height) was filled with 5.0 kg of soil (taken from the experimental field), under a natural condition in a net house. Distilled water was irrigated twice a week until measurement.

Salinity treatment was applied to seedlings with 2 months of age (germination period was counted). In salt-treated groups, different NaCl solutions (50, 100, 200, 300, or 400 mM) were irrigated to the plants every 3 days. Each pot irrigated with 50 ml per time. Distilled water served as the control treatment (CK1).

According to the preliminary experiment, no visible damage on the plant was observed with 50-,100-, 200-, or 300-mM salt stress applied for 20 days. However, 400-mM salt treatment for a week caused plant wilting. Salt stress of 500 mM for 24 h led to plant death. So, 300-mM salt stress was set as CK2 in a salt + SA test. In salt + SA groups, 0.05- and 0.1-mM SA solutions were irrigated to the evaluated plants treated with 300-mM salt solution. A 50-ml (salt) + 30-ml (SA) solution was given every 3 days time. The treatments continued to the end of the experiment. The final harvest occurred after 15 days of salt treatment. Plants were then separated in shoots, stems, and roots and oven dried at 60 °C.

Daily average growth

Height of the aerial part, as the measurement of daily average growth, was measured at 0 (determination was conducted after treatment), 3, 6, 9, 12, and 15 days of treatment, then the average growth was calculated. Daily growth rate was calculated as follows:

$$ \mathrm{Daily}\;\mathrm{average}\;\mathrm{growth}=\left({H}_n-{H_n}_{-3}\right)/3 $$
H n :

Plant height on day n

H n–3 :

Plant height on day (n–3).

Activity of antioxidase

For enzyme extraction, 0.5 g of fresh samples were ground to a fine powder with liquid nitrogen and extracted in 5 ml of 50-mM sodium phosphate buffer (pH 7.8). The extracts were centrifuged at 10,000 × g for 20 min at 4 °C. Then, the supernatant was used as the crude extract enzyme preparation for the activity of antioxidant enzyme measurement.

The activity of superoxide dismutase (SOD) was assayed according to Li (2000). A 3-ml reaction mixture contained 50-mM phosphate buffer (pH 7.8), 13-mM methionine, and 2-μM riboflavin. Nitro blue tetrazolium 75 μM, EDTA10 μM, and 0.05 ml of enzyme extract were added to the reaction mixture. The reaction was started by exposing the mixture to a cool white fluorescent light at an intensity of 4,000 lux for 15 min. One unit of SOD activity was defined as the amount of enzyme required to cause 50 % inhibition of the rate of nitro blue tetrazolium chloride reduction, and the specific enzyme activity was expressed as units per milligram of protein.

The activity of peroxidase (POD) was assayed according to Li (2000). The reaction mixture contained 50-mM sodium phosphate buffer (pH 6.0), 2 % H202, 0.05-M guaiacol, and 0.1-mL enzyme extract in a final volume of 5 mL. The reaction was started by the addition of enzyme extract. POD activity was expressed as units per mg of protein.

Catalase (CAT) activity was assayed by the method of Gao (2006). The final volume of the reaction mixture contained 50-mM sodium phosphate buffer (pH 7.8) and 0.1-M H202. The reaction was started by adding 0.2 ml of leaf crude extract to this solution, and the activity was calculated as units (umol H202 consumed per min) per milligram of protein.

Proline content

Proline content was estimated by the method of Demiral and Türkan (2005) with a little modification. Leaf samples 0.5 g were added into 3 % (w/v) sulfosalicylic acid in a test tube (5 ml). After plugging the tube, the mixture was heated at 100 °C for 15 min in a water bath and filtrated. The filtrate (2 ml) was heated at 100 °C for 15 min in a water bath after adding acidic ninhydrin (3 ml) with glacial acetic acid (2 ml) and then stopped at room temperature (25 °C). The mixture was extracted with toluene. Absorbance of fraction was read at 520 nm (MAPADA UV-1600PC spectrophotometer). Proline content was calculated using a calibration curve and expressed as microgram proline per gram FW.

Determination of mineral elements

The samples of different plant parts were digested with a mixture of HNO3 and HClO4 (4:1, v/v). The resultant solution was diluted to a volume of 50 mL with deionised water (Xu et al. 2009). Blank solutions were prepared in the same manner as the samples. Then all the prepared solution was analyzed for the accumulation of elements by atomic absorption spectrophotometer (AA-6300, SHIMADZU, Japan).

Data analysis

All experiments were repeated at least three times and averaged (±SE). The data were analyzed by Duncan’s multiple range test and t test at P < 0.05 using the PC SPSS 20.0. Significances between means were assessed using the SPSS ANOVA procedure. Figures 1, 2, 3, and 4 were completed through Origin 8.0.

Fig 1
figure 1

Activity of SOD in Artemisia annua L. under different stressed conditions (0, 50, 100, 200, 300, or 400 mM of NaCl) over the experimental period. CK 1 (0 mM of NaCl) means the untreated group, CK 2 (300 mM of NaCl) means the control in SA + NaCl treatment groups. Each value is the mean of three replicates. Vertical bars indicate the mean ± SE

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Fig 2
figure 2

Activity of POD in Artemisia annua L. under different stressed conditions (0, 50, 100, 200, 300, or 400 mM of NaCl) over the experimental period. CK 1 (0 mM of NaCl) means the untreated group, CK 2 (300 mM of NaCl) means the control in SA + NaCl treatment groups. Each value is the mean of three replicates. Vertical bars indicate the mean ± SE

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Results

Plant daily average growth rate

Daily average growth rate for A. annua under different salt concentrations and SA treatments was shown in Table 1. In salt-treated groups, average growth rate of the CK1 was higher than that of the treated groups during the measurement. A negative relationship was detected between the growth parameter and the increasing salt concentration. The highest inhibitory effect was shown in an average growth rate which decreased from 0.83 (CK1) to 0.12 (400 mM) during 13–15 days. Average growth rate of salt-treated groups consistently reduced with days. In salt + SA-treated groups, exogenous SA had a positive effect on a plant’s daily average growth rate when compared with CK2 (300-mM NaCl) and especially at 0.05 mM. Obviously, we found that the daily growth rate increased from 0.25(CK2) to 1.08 (0.05-mM SA) after 15 days of treatment.

Table 1 Effect of salicylic acid and salt stress on growth rate of A. annua (n = 4)
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Overall, salt stress inhibited the growth of A. annua, and inhibition intensity was positively governed by the increasing salt concentration. Salicylic acid had some alleviation effects on A. annua’s growth inhibition.

Antioxidase activity

Activity of SOD in A. annua exposed to salt- and salt + SA-treated plants were exhibited in Fig. 1. SOD activity first increased and then decreased in the whole experimental process, of which the maximum activity was detected on the ninth treatment day. Enzyme activity was higher in all treated groups than in CK1. In the salt-treated groups, the SOD activity depended on salt dosage, in which the highest SOD activity was induced by 100-mM salt treatment, followed by that in 300, 400, and 50 mM. In the salt + SA-treated groups, 0.05-mM SA effectively stimulated SOD activity, whereas 0.1-mM SA had no obvious effect on enzyme activity.

According to Fig. 2, POD activity was increasing in the experiment and rose to the highest at the end. There was a higher level of POD activity in treated plants than the CK1. And it was observed that POD activity was higher in the salt groups than in the salt + SA groups. In salt groups, POD activity reached the highest in a 200-mM salt treatment, next was 100 or 300 mM. Enzyme activity showed no visible difference between 100- and 300-mM treatments, however, slightly higher than that in a 50-mM salt treatment. In the salt + SA groups, the application of 0.05-mM SA stimulated a higher POD activity than both 0.01-mM SA and CK2.

CAT activity was depicted in Fig. 3. In contrast to POD, CAT activity sharply increased after 3 days of treatment and reached the maximum during 6 to 9 days, whereas it showed a continuous decline from 9 to 15 days. Activity of CAT was much higher in the treated plants than in the CK1. In salt groups, the highest CAT activity was observed at 300-mM salt treatment. In the salt + SA groups, the activity of CAT in salt + SA treatment was far lower than CK2.

Fig 3
figure 3

Activity of CAT in Artemisia annua L. under different stressed conditions (0, 50, 100, 200, 300, or 400 mM of NaCl) over the experimental period. CK 1 (0 mM of NaCl) means the untreated group, CK 2 (300 mM of NaCl) means the control in SA + NaCl treatment groups. Each value is the mean of three replicates. Vertical bars indicate the mean ± SE

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Proline

The result pertaining to the effect of salt stress on proline level was depicted in Fig. 4. It was clear that the content of free proline gradually increased and reached the maximum at the last 3 days. Salt stress stimulated production of proline. In salt groups, the maximum proline content was induced by 200-mM salt stress. In SA + salt groups, proline concentration was enhanced with the application of 0.05-mM exogenous SA when compared with the CK2, whereas 0.1-mM SA had an inhibitory effect on the proline content.

Fig 4
figure 4

Proline level in Artemisia annua L. under different stressed conditions (0, 50, 100, 200, 300, or 400 mM of NaCl) over the experimental period. CK 1 (0 mM of NaCl) means the untreated group, CK 2 (300 mM NaCl) means the control in SA + NaCl treatment groups. Each value is the mean of three replicates. Vertical bars indicate the mean ± SE

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Mineral element

After 15 days of salt stress, the distribution of a mineral element in the plant organs was shown in Table 2. Na+ was mainly accumulated in the root, of which a small amount was distributed in stem and leaf. While a significant increase of Na+ concentrations in the root of the A. annua was stimulated by salt treatment, this excessive Na+ absorption to some extent was inhibited with the application of SA. K+ was mainly accumulated in leaf, which is followed by root and stem. The highest K+ concentration among all the salt-treated groups was induced by 200-mM salt stress. However, K+ content of SA treatment groups reduced in comparison with CK2. Ca2+ concentration was highest in the root; leaf and stem followed. Salt treatment led to a drop of Ca2+ concentration. Content of Ca2+ in A. annua under 200-mM salt stress was higher than that of other treatments (excluding the CK1) in each organ. However, compared with CK2, a slight decrease of Ca2+ content occurred when SA was applied to the plant. Mg2+ was mainly accumulated in stem. Mg2+ content of the treated plants was slightly higher than that of the CK1. Salt stress had a positive effect on the accumulation of Mg2+, especially at 300-mM salt concentration in stems. Treatment of salt + SA failed to promote Mg2+ accumulation compared with CK2.

Table 2 Accumulation of element in A. annua response to salt stress (n = 3)
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Discussion

Salinity always imposes threats such as low water potentials, ion toxicities, and nutrient deficiencies (Amor et al. 2005; Gautam and Singh 2009). These threats would expose additional stress on physiological and biochemical processes in plants, further inhibiting growth. In our studies, daily average growth of A. annua was hindered slowly with the increasing salt concentration. A severe growth reduction derived from salt stress was observed. It was similar to those found in linseed and cucumber (Khan et al. 2010; Wei et al. 2004). Present study suggested that SA had more or less restoration effect on salinity damage, it could protect membrane structure from salt damage; integrity of membrane not only facilitated the photosynthesis process to continue normally but also segregated excess anion and cation into vacuolar compartment and thus contribute to ion homeostasis (Guo et al. 2011; Singh and Gautam 2013; Stevens et al. 2006). Our results showed that the deleterious effect of salinity on plant growth was effectively alleviated by exogenous SA. Moreover, the degree of alleviation was strengthened by prolonged time. It means that SA had a good effect in protecting the plant from salt injury.

Plants subjected to salt stress would produce reactive oxygen species (ROS) which stimulate nonspecific oxidation and oxidative damage. SOD, POD, and CAT are among the major antioxidant enzymes involved in the scavenging of ROS (Amor et al. 2005; Tang et al. 2007). Our results showed that increasing salt concentrations led to a significant change in SOD, POD, and CAT activities in evaluated plants. SOD kept a relatively high activity in the whole experiment process, which indicated that SOD played a critical role in scavenging ROS in A. annua under salt stress, and consolidated with the existing viewpoint which stated that SOD acted as a major scavenger in protecting the plant from ROS (Tang et al. 2007). SOD effectively scavenged ROS at first that its activity recovered to a normal level finally. In contrast to POD, CAT fastly showed a higher activity level during the experiment. POD was activated after the CAT activity reached to a maximum. It may indicate that CAT was more sensitive to salinity in A. annua when compared with POD. Moreover, CAT and POD concertedly acted as hydrogen peroxide scavengers during the experimental process.

Exogenous SA produced an inconformity effect on different antioxidant enzymes. SOD and POD activities were stimulated by SA at 0.05 mM, inversely was inhibit by SA at 0.1 mM. This suggested that the effect of SA on antioxidase activity in a salt-stressed plant was related to its concentration. It was worth mentioning that CAT activity was inhibited by SA at both concentrations. Kang et al. (2004) and Sawada et al. (2008) reported that SA-binding protein (SARP) and CAT were highly homologous, and CAT activity would be reduced when SA functioned on SARP. As a result, a portion of hydrogen peroxide not cleared by CAT acted as a second messenger to further stimulate the defense reaction. Effect of SA on antioxidase in this study was in good agreement with the results of the present investigation on the response of antioxidase in Ammopiptanthus mongolicus, and soybean survived to salt with SA treatment (Liu et al. 2006; Kumara et al. 2010). Suppression of SA for CAT activity gave us some hints that if we can try to modify the three-dimensional structure of SA in order to promote, plant acquired a better antioxidant effect. This idea needs further experimental verification.

As a physiologically compatible solute, proline increased as needed to maintain a favorable osmotic potential between the cell and its surroundings (Demiral and Türkan 2005; Gautam and Singh 2009). Recent studies had some debate on whether proline accumulation enhanced the salt tolerance of plants or proline accumulation was the result of osmotic stress (Wang and Han 2009). In our current study, content of proline in both salt and salt + SA groups was much higher than the CK1. Proline accumulation showed a gradual increase with increasing stress level, especially in the long time of treatment. This indicated that proline accumulation was the result of salt tolerance. Our results strengthened the latter the debate. As compared with CK2, proline content slightly increased with 0.05-mM SA treatment, but greatly decreased with 0.1-mM SA. Probably, SA concentration also had a relationship with proline level in salt-stressed plants. Specific reason remains to be further researched.

An important strategy for salinity tolerance in highly evolved plants was restricting Na+ mobility to prevent Na+ from accumulating in the stem and leaf (Lõpez-Aguilar et al. 2003). This was demonstrated in this paper with the results that Na+ was mainly distributed in the root, and only a small portion was allocated in stem and leaf. K+ content in roots of treated plants was lower than in the CK1, which indicated that the uptake of K+ reduced under a salt stress environment. Excess accumulation of Na+ would limit the absorption of K+ (Khan et al. 2000; Zou et al. 2011). So the ratio of sodium to potassium (Na+/K+) might be used as an indicator of salt tolerance. In this study, Na+/K+ (Table 2) gradually increased with increasing salt concentration in each organ of the evaluated plants; a strong negative correlation between K+ over Na+ uptake in A. annua under salinity was observed. Our results were consistent with previous studies (El-Tayeb 2005; Guo et al. 2011; He and Zhu 2008). Obviously, increased ratio of Na+ and K+ was the result of excessive Na+ uptake. Excessive Na+ accumulation in a cell and its surroundings would impair membrane integrity, in which a major membrane channel protein is distributed. Absorption of Ca2+ would be very difficult to complete without membrane channel proteins assisted. On the other hand, Ca2+ appeared to be readily displaced by excessive Na+ from its membrane-binding sites (Cachorro et al. 1994). Therefore, maybe the absorption of excessive Na+ leads to a Ca2+ content decrease in the root. Results on the accumulation of Mg2+ in the plant that suffered salt stress were not consistent. However, SA was consistently helpful to Mg2+ accumulation (Gunes et al. 2007; He and Zhu 2008). The specific reason needs further research.

Conclusion

The study revealed that salt stress promoted inhibitory effects on the growth of A. annua plants. Salt stress stimulated the activity of antioxidase, production of proline, and absorption of Na+, Mg2+, and K+ while inhibiting the uptake of Ca2+. Exogenous application of SA alleviated the growth inhibition and oxidative damage result from salt stress in A. annua and effectively inhibited the excessive absorption of Na+. All these indicated that SA could be used as a potential growth regulator to improve A. annua growth and nutrient utilization under salt stress. These would provide some practical basis for a wide cultivation of A. annua.