In the Qinghai-Tibet Plateau, both the large daily temperature difference and soil salinization make plants susceptible to abiotic stresses such as freeze-thaw and salinity. Meanwhile, crops in this area could be subjected to the influence of artemisinin, an allelochemical exuded by Artemisia annua. In the context of freeze-thaw and salinity stresses, artemisinin was induced as an allelopathy stress factor to explore the physiological response of highland barley, including the relative electrical conductivity (RC), soluble protein (SP) content, malondialdehyde (MDA) content, antioxidant enzyme activity, and water use efficiency (WUE).There data suggested that artemisinin weakened the self-osmotic adjustment ability of seedlings, reducing the SOD activity in scavenging efficiency of reactive oxygen species, then causing oxidative damage to cell membrane of seedlings, which significantly increases the content of RC and MDA. Artemisinin stress can reduce the WUE of seedlings and weaken the photosynthesis intensity of seedlings as well. In a word, salinity stress and artemisinin respectively showed a synergistic compound relationship with freeze-thaw stress,
Research Article
Barley Seedlings in Response to Salinity and Artemisinin Stress in the Present of Freeze-Thaw Stress
https://doi.org/10.21203/rs.3.rs-1034052/v2
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Allelochemicals
Photosynthesis
Qinghai-Tibet Plateau
Soil salinization
Physiological effects
freeze-thaw
Known as the "Roof of the World", the Qinghai-Tibet Plateau is very sensitive to climate change. In addition, complex geographical and climatic conditions make seasonal frozen soil being widely distributed on the plateau (Wang et al, 2019), resulted from which, the freeze-thaw stress not only affects the function of cell membranes and osmotic adjustment of plant (Bertrand et al, 2016; Min et al, 2020),but also affect the photosynthesis of the above-ground parts of plants through changing soil nutrients, resulting in a decline in vegetation productivity (Domisch et al, 2019; Arfan et al, 2019). The evaporation in the Qinghai-Tibet Plateau generally exceeds the actual precipitation, accelerating the accumulation of salt to the soil surface (Guo 2021),besides, improper agricultural production measures like an increasing amount of chemical fertilizers usage have resulted in the secondary salinization of soil more and more serious (Che et al, 2021).It has been reported that massive salt in the soil could easily cause osmotic stress, ion toxicity and metabolic hindrance to plants, which therefore affects mostly important life processes of plants (Zhang et al, 2021). Recently, the artemisinin secreted by Artemisia annua that is one of the wild medicinal resources in the Qinghai-Tibet area (Ye et al, 2014) would cause allelopathy to the plants growing around it (Herrmann et al, 2013; Hussain and Reigosa, 2014). Studies have shown that a higher concentration of artemisinin solution can inhibit the germination of various crop seeds, reducing photosynthetic rate, root vitality and protective enzyme activities at the seedling stage of crops (Yan et al, 2018).
Highland barley (Hordeum vulgare Linn. var. nudum Hook.f.) is a cereal crop of the gramineous barley genus, and is the main crop in Tibet and Qinghai (Qian et al, 2019). In this paper, highland barley seeds of variety Beiqing No.3 were obtained commercially in the market from an agricultural seed company Sichuan Tianlu Agricultural Science and Technology Development CO.,LTD and used as the experimental material subjected to the combined stresses of salinity salt and artemisinin. Artificially simulated freeze-thaw experiment was carried out to investigate the physiological responses of barley to freeze-thaw stress, salinity stress and allelopathic stress through measuring relative electrical conductivity, soluble protein content, malondialdehyde content, antioxidant enzyme activity and water use efficiency.
2.1 Plant culture and compound treatments of salinity and artemisinin
The full-grained highland barley seeds were selected and disinfected with 0.1% KMnO4 solution for 2h, after which the seeds were rinsed with deionized water until the water becoming clear. Then we spread 120 seeds evenly on each of 8 culture dishes randomly named SAF, SOF, OAF, OOF, SAO, SOO, OAO and OOO (Table 1). The artemisinin powder (purity ≥ 98%) was purchased from Shaanxi Sciphar Natural Products Co., Ltd. Dissolve 20mg artemisinin powder in 1ml acetone to prepare stock solution, then stock solution was diluted to get a concentration of 20 mg/L artemisinin solution (A solution) with 1/2 Hoagland nutrient solution (Yan et al, 2015). NaHCO3 was added into 1/2 Hoagland nutrient solution and 20 mg/L artemisinin solution respectively, to get a 60 mM salinity stress solution (S solution) and 60 mM compound stress solution of salinity and artemisinin (S-A solution) prepared.
500ml S-A solution was added to the cultivated dishes of Groups SAF and SAO at the same time, and 500ml S solution for Groups SOF and SOO, 500ml A solution for Groups OAF and OAO, 500ml 1/2 Hoagland nutrient solution for Groups OOF and OOO. 8 culture plates were placed into MGC-450BP light incubator (Shanghai Yiheng Scientific Instruments Co., Ltd) for germination. The cultivated conditions were set as 12 h light (25 ℃) and 12 h non-light (15 ℃). Daily watering (50 ml) was necessary during the cultivation.
|
SAF |
SOF |
OAF |
OOF |
SAO |
SOO |
OAO |
OOO |
|
|---|---|---|---|---|---|---|---|---|
|
Salinity |
+ |
+ |
- |
- |
+ |
+ |
- |
- |
|
Artemisinin |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
|
Freeze-thaw |
+ |
+ |
+ |
+ |
- |
- |
- |
- |
| + add stress, - no stress | ||||||||
2.2 Freeze-thaw stress treatment and sampling
After having grown for 8d, the seedlings of Groups SAF, SOF, OAF and OOF were put into BPHJ-120A high-low-temperature test chamber (Shanghai Yiheng Scientific Instruments Co., Ltd) to perform a freeze-thaw cycle for a period of 14 h, while other cultivated dishes of Groups SAO, SOO, OAO and OOO were maintained in light incubator under previous culture conditions. Initially, the cultivated dishes were placed in the chamber at 15 ℃ that closed to room temperature at night. Controlled precisely by program, the temperature decreased to -5℃ steadily at a speed around 0.04 ℃/min, and then the temperature increased from -5 to 10 ℃ at a speed around 0.04 ℃/min. After the freeze-thaw cycle being started, five parallel samples were taken at 2h, 8h and 14h from 8 cultivated dishes at random according to the required amount of the measurement, the corresponding sampling temperature was 10, -5, 10℃ respectively, recorded as T1, T2, and T3.All the samples were firstly wrapped up with tin foil paper, secondly fixed in liquid nitrogen immediately for 50 s and finally put into the ultra-low-temperature freezer at -80℃ for storage in order to measure the content of MDA and soluble protein, SOD and POD activity. At the same time, fresh leaves were taken to determinate RC and WUE.
2.3 Analysis
2.3.1 Relative electrical conductivity (RC)
The relative electrical conductivity (RC) of leaves was measured before and after heat treatment to assess cell membrane damage, as described by He et al. with modifications (He et al, 2018). In brief, sampled leaves (0.1g) were washed using deionized water for 3 times, cut into 0.5-1cm pieces, and immersed in 10ml deionized water for 12h at room temperature. Then, RC of the solution was measured using a conductivity meter (DDS-307, INESA Scientific Instrument Co., Ltd., Shanghai, China) and recorded as S1. After boiling the leaf samples for 30 min, RC of the solution at room temperature was measured again and recorded as S2. A RC was calculated according toformula (1).
2.3.2 Soluble protein (SP)
The soluble protein content was determined by the Coomassie brilliant blue method (Bradford 1976). For each replicate, 0.1 g leaves were homogenized with 5 ml distilled water, and the mixture was centrifuged with a TDL-40B centrifuge (Anting Scientific Instrument Factory, Shanghai) at a speed of 3000 r/min for 10 min. A 1 ml sample of the supernatant was diluted in 5 times with 4 ml distilled water, of which 1 ml diluted supernatant was taken into a test tube with 5 ml of Coomassie brilliant blue solution being added. After the mixed solution being shaken and placed for 2 min, the absorbance at 595 nm was measured with a UV-6100 UV–visible spectrophotometer (Metash Co. Ltd) and translated into protein content using a calibration curve constructed with bovine serum albumin as standard.
2.3.3 Malondialdehyde (MDA)
Malondialdehyde (MDA) content was determined by the thiobarbituric acid method.(Gong et al, 2020)For each replicate, around 0.5 g leaves were homogenized with 5 ml 10% trichloroacetic acid (TCA) solution, and the mixture was centrifuged at a speed of 4000 r/min for 10 min. Then 2 ml of the supernatant was taken and fixed with 2 ml 0.6% thiobarbituric acid (TBA) solution. Mixtures was bathed in 99 ℃ water for 15 min, then cooled quickly in 5 min and centrifuged again at a speed of 4000 r/min for 10 min with a TDL-40B centrifuge (Anting Scientific Instrument Factory, Shanghai). The absorbance of supernatant was measured at 532 nm, 600 nm, and 450 nm with a UV-6100 UV–visible spectrophotometer (Metash Co. Ltd). The MDA concentration and MDA content were calculated according to formulas (2) and (3).
where:
• D450, D532, D600 are the absorbance at 450nm, 532nm and 600nm, respectively.
• cMDA is MDA concentration (μmol/L);
• VT is the volume of TCA solution (ml);
• FW is the fresh weight of seedlings (g).
2.3.4 Superoxide dismutase (SOD) and peroxidase (POD) activities
The activities of SOD and POD were determined with the SOD and POD kits provided by Nanjing Jiancheng Biological Institute. A parallel sample (around 0.25 g) were randomly taken and homogenized with 5 ml phosphate buffer on ice. After centrifugations at a speed of 3500 r/min and 3500 r/min with a TDL-40B centrifuge (Anting Scientific Instrument Factory, Shanghai), respectively for 10 min, the supernatant was used for following measurements according to instructions of kits.
2.3.5 Water use efficiency (WUE)
The CIRAS-3 portable photosynthesis system(PP SYSTEMS, American) was used to determine the water use efficiency (WUE) in the barley seedling leaves. The CO2 buffer bottle is placed in a ventilated place when the photosynthesis measurement experiments were performed. The instrument is provided with a built-in LED light source. The setting parameter is PLC3 Universal Leaf Cuvette (7×25 mm window), 80% for H2O Reference, 300 µmol·m-2· s-1 LED for Light Source, and 90, 0, 5, and 5 respectively for the Red-Green-Blue-White parameters in RGBW Control, besides, the temperature is controlled to 25±2℃.
2.4 Statistical analysis
The experiments were repeated five times, and the data were expressed as mean ± standard error (SE) (n=5), which statistically performed with R 3.3.1 statistical software (R Foundation for Statistical Computing, Vienna, Austria) for one-way analysis of variance (ANOVA) and Multivariate ANOVA. When the variables were uniform, the significance analysis of data was analyzed using Duncan model, otherwise using Games-Howell model (Warner 2013). All results were shown in bars in figures plotted by Origin 8.0 software. Different letters presented in figures indicated significant differences between different treatment groups at the same time.
3.1 Effect on the relative electrical conductivity (RC) of seedlings
It can be seen from Fig. 1a that exposure of highland barley to stresses caused a stress-dependent increase in relative electrical conductivity (RC) (P<0.05),and the maximum increase was observed under the compound stresses of salinity, artemisinin and freeze-thaw. Exposure of barley leaves to freeze-thaw stress exacerbated the effects and the RC in these plants at T3 represented 83.34% (Group SAF), 102.51% (Group SOF), 64.17% (Group OAF) and 11.88% (Group OOF) higher than that at T1 (P <0.05), while the treatment groups that not subjected to freeze-thaw stress represented no significant change in T1-T3. Furthermore, freeze-thaw stress significantly increased the RC in barley leaves of the four treatment groups (SA*, SO*, OA* and OO*) after treatment with freeze-thaw (Status **F) as compared to Status **O (Fig. 1b).The results above indicated that freeze-thaw, salinity and artemisinin stress had independent effects on RC, but had a significant synergistic relationship with each other.
3.2 Effect on the soluble protein (SP) content of seedlings
Figure 2a depicted that, under non-freeze-thaw conditions, artemisinin stress decreased soluble protein (SP) content as compared to control, however, there were no differences in SP content between control and treated plants under salinity stress. Simultaneous with the above changes, the SP content of leaves increased initially and then decreased in the groups treated with freeze-thaw stress, among which, SP content of Group SAF and SOF reduced 36.1% and 15.6% after treatments with freeze-thaw respectively, while Group OAF and OOF increased 47.7% and 102.9% respectively. It can be observed from Fig. 2b that the SP content of leaves in Group SA* decreased, while that in Group SO*, OA* and OO* increased, indicating that freeze-thaw had a significant synergistic relationship with artemisinin stress and salinity stress respectively (P <0.05), and that the group under salinity and artemisinin conditions could not synthesize more SP by itself to resist the adversity induced by freeze-thaw stress.
3.3 Effect on malondialdehyde (MDA) content of seedlings
The malondialdehyde (MDA) content of leaves in groups treated with stressincreased by 16.29% ~ 82.62% compared to the control (Fig. 3a). Under non-freeze-thaw conditions, no significant change of MDA content in barley leaves was observed within 14-hour cultivation, while artemisinin stress increased the MDA content of leaves (P <0.05). Meanwhile, the MDA content of the barley seedlings at lowest temperature (T2) in Groups SAF, SOF, OAF and OOF respectively represented 23.8%, 17.9%, 19.7% and 34.7% higher than that at the initial temperature (T1) (P <0.05). Fig. 3b suggested that the slope of Group SO* was the same as that of Group OO*,and the similar relationship was observed between Groups SA* and OA*.That is, the freeze-thaw stress and salinity stress are independent regardless of whether the artemisinin stress existed or not.
3.4 Effect on superoxide dismutase (SOD) activity of seedlings
As shown in Fig. 4a, at T1, freeze-thaw treatments reduced the SOD activity of the barley seedling leaves compared to the control(Group **O). During the freeze-thaw cycle, the SOD activity in the barley seedlings leaves increased by 10.0% (SAF), 27.0% (SOF), 11.3% (OAF) and 18.7% (OOF). Fig. 4b depicted the increase in SOD activity of Group SA*, SO*, OA* and OO*, indicating that freeze-thaw stress had an interaction relationship with artemisinin stress (P <0.05). In addition, the increase in SOD activity of Group SO* after freeze-thaw treatment was more than that of Group OO*, while the increase in SOD activity of Group SA* after freeze-thaw treatment was less than that of Group OA*, which demonstrated that under the artemisinin condition, freeze-thaw stress had an interaction relationship with salinity stress as well (P<0.05).
3.5 Effect on peroxidase (POD) activity of seedlings
There was no significant change in POD activity of leaves in groups growing under non-freeze-thaw conditions within T1-T3 (Fig. 5a). However, the effects of freeze-thaw on POD activity of the barley seedling leaves were evident after 14-hour exposure, which was represented as a downward trend during freeze-thaw cycle. As can be observed in Fig. 5b, after 14-hour exposure of freeze-thaw stress, except for the increase in POD activity in the SA* treatment group, the POD activity in SO*, OA* and OO* treatments all decreased to varying degrees,proving that freeze-thaw stress had a very significant interaction relationship with artemisinin stress and salinity stress respectively (P <0.01).
3.6 Effect on water use efficiency (WUE) of seedlings
The water use efficiency (WUE) of barley seedling leaves growing in the presence of freeze-thaw stress decreased significantly during the thawing process (T2-T3) and was much lower than that growing under non-freeze-thaw conditions (Fig. 6a). The maximum reduction of WUE was recorded in barley seedlings of Group SAF (-189.9%), followed by Group SOF (-154.9%) and Group OAF (-154.1%), and the Group OOF decreased the least by -127.6% with respect to the WUE at T1. We could see from Fig. 6b that the decreasing slope of Group OA* was the same as Group OO*, which might indicate that the freeze-thaw stress and the artemisinin stress are independent in the conditions of non-salinity stress. On the contrary, the decreasing slope of Group SA* differed from that of Group SO*, indicating that the freeze-thaw stress had significant synergistic relationship when exposed tosalinity stress (P <0.05). Similarly, the freeze-thaw stress had a synergistic relationship with salinity stress no matter whether artemisinin stress was applied or not (P<0.05), which could be demonstrated in Fig. 6b.
It is well-documented that the application of compounds with allelopathic effectscould result in the damage to the function of plant cell membranes (Romagni et al, 2000). Induced by adversity stress, excessive reactive oxygen species (ROS) in cells could lead to membrane peroxidation, resulting from which, the structure of plant cell membranes was destroyed, leading to an increasing RC (Wang et al, 2021). RC is characterized by the selective permeability function of cell membranes and, for this reason, RC is highly susceptible to environmental stresses. In this present study, single or combined stress of artemisinin, salinity and freeze-thaw increased the RC of barley seedlings compared to the control, which suggested that, to a certain extent, the cell membrane of leaves may have been destroyed under environmental stresses. Therefore, the reason for increased RC was the electrolyte extravasation due to the loss of selective permeability caused by damaged cell membrane. This may attribute to the accumulation of excessive ROS in the cells (Apel and Hirt, 2004). The maximum increase of RC was observed in Group SA* that followed by Group OA*, Group SO* and Group OO*, proving that the combined stresses of salinity and artemisinin reduced the regulating ability of plants and that salinity stress was more harmful to cells than artemisinin stress. Additionally, MDA increased when the cell membrane was damaged by ROS, and its content could reflect the severity of the stress which the plant subjected to (Karagoz et al, 2018). Previous researches proved that MDA content would increase in those plants exposed to freeze-thaw stress (Balestrasse et al, 2010), and similar results were observed in our study. During the freeze-thaw stress cycle, compared with the single freeze-thaw group, both salinity and artemisinin stress significantly increased the MDA content in the barley leaves, indicating that the two stresses further exacerbatethe damage (Babula et al, 2009).
Allelochemicals appear to alter a variety of physiological processes and it is difficult to separate the primary from the secondary effects. Several action modes have been suggested, including direct inhibition of protein synthesis (Yan et al, 2018) and degradation of macromolecular proteins (Liu et al, 2008). From the results, it is clear that artemisinin stress reduced the SP content of barley leaves, attributing to allelochemicals initially reduce the incorporation of certain amino acids that are raw materials of protein synthesis, consequently, the rate of protein synthesis reduced (Baziramakenga et al, 1997). Under freeze-thaw condition, the SP content of the seedlings increases significantly as the temperature dropping down, which is consistent with previous studies, in which researchers demonstrated that the accumulation of SP induced by the decreasing temperature might reduce the osmotic potential of cells (Airaki et al, 2012), improving the water retention capacity, and eventually, the damage of freeze-thaw stress to plants was relieved (Bao et al, 2019b). The SP content of seedling leaves in the present of salinity stress (SO*) increased more, while that of leaves in the present of artemisinin stress (OA*) increased less compared to control (OO*), indicating that the salinity stress may improve the resistance of highland barley to freeze-thaw stress to a certain extent, while artemisinin stress might make highland barley more sensitive to freeze-thaw stress, and the combined stress of salinity and artemisinin may weaken the osmotic adjustment ability of highland barley.
Changes in the antioxidant enzymes activity are good indicators of improving their resistance to adversity. In the present study, it was observed that SOD activity of the leaves in groups in the exposure of freeze-thaw stress was significantly enhanced (Meng et al, 2016). Plants can produce a variety of by-products of aerobic metabolism in the process of physiological metabolism. When entering the plant, oxygen initially produces O2-, which would be converted into ROS including H2O2 and -OH. It has been proved that excessive ROS can induce cell apoptosis (Gechev et al, 2006). In the early stage of stress, the antioxidant enzymes activity of wheat seedlings increased to resist the adversity, however, as the stress exacerbates, the antioxidant enzymes activities would decrease then, aggravating the membrane lipid peroxidation (Bao et al, 2019a). Similarly, compared with the control, the SOD activity of the highland barley seedlings in the present of salinity and artemisinin decreased, and POD activity increased. Continuous application of salinity and artemisinin reduced SOD enzyme activity, leading to the accumulation of ROS and the increasing severity of membrane lipid peroxidation, and consequently, increasing the MDA content. After freeze-thaw stress being added, the SOD activity of highland barley seedlings increased, and the POD activity decreased, which might be SOD enzyme activity plays an important role in improving the tolerance of barley to freeze-thaw (Bian et al, 2020).
Water is one of the most important materials for photosynthesis, and leaf WUE described the relationship between the formation of organic matter and the water usage during plants growth (Liang et al, 2013). Under non-freeze-thaw conditions, no changes were observed in the WUE of barley leaves, and there was no significant difference between those groups. The results above indicated that a normal WUE of barley still maintained although it is under the stress of salinity and artemisinin. On the contrary to the former results, the WUE of barley in freeze-thaw treatment groups initially decreased little and subsequently dropped to a negative value during the thawing stage. Perhaps because the barley slowly adapted to adversity as the temperature dropped and the low temperature inhibited the synthesis of chlorophyll, which effected and reduced the photosynthetic rate. This was confirmed in Bao's research (Bao et al, 2020). Subsequently, in the process of thawing, the WUE represented a negative value, which might due to the fact that the newly synthesized organic substances in photosynthesis can no longer meet the energy demand of plant respiration under the low temperature environment.
In summary, as an important crop on the Qinghai-Tibet Plateau, barley has equipped with great resistance to the freeze-thaw environment within the long-term evolution. However, improper agricultural production measures have led to increasingly serious salinization. Besides, the artemisinin, an allelochemical, is widely distributed on the Qinghai-Tibet Plateau. These abiotic stresses mentioned above would lead to complex physiological changes in plants. In the present study, RC and MDA content in barley leaves were increased due to the application of salinity, artemisinin and freeze-thaw stress, among which, compound stresses caused the most serious damage to plant cell membrane permeability. Moreover, the SP content of barley present an initial increasing and a subsequent decline during freeze-thaw stress, suggesting that the exposure of barley to artemisinin stress inhibited the self-osmotic regulation when subjected to freeze-thaw stress at the same time. When it comes to antioxidant enzymes activity and WUE, freeze-thaw had significant synergistic relationship with artemisinin stress and salinity stress respectively. On the whole, freeze-thaw stress presented a synergistic stress relationship with salinity and artemisinin stress. Though the resistance characteristics of plants under one freeze-thaw cycle were studied in this paper, in view of multiple freeze-thaw cycles in nature, one important future direction of physiological responses to freeze-thaw stress is studying the different resistance characteristics of plant between under one freeze-thaw cycle and under multiple freeze-thaw cycles.
ACKNOWLEDGMENTS
This work was sponsored by the National Natural Science Foundation of China (Grant Nos. 31772669 and 32071874)，Key Projects of Science and Technology Development Plan of Jilin Province (Grant No.20210203001SF), Interdisciplinary Project of Jilin University（Grant No. JLUXKJC2020107）and the 111 Project (B16020)
FUNDING
This work was sponsored by the National Natural Science Foundation of China (Grant Nos. 31772669 and 32071874)，Key Projects of Science and Technology Development Plan of Jilin Province (Grant No.20210203001SF), Interdisciplinary Project of Jilin University（Grant No. JLUXKJC2020107）and the 111 Project (B16020)
COMPETING INTERESTS
The authors have no relevant financial or non-financial interests to disclose.
AUTHOR CONTRIBUTIONS
When writing statement, Zhang Wei and Tang Wenyi designed research. Zhang Wei, Tang Wenyi, Xiao Jing, Dai Gejun, Liu Jiapeng and Wang Zhao conducted experiments. Zhang Wei and Dai Gejun wrote the main manuscript and Xiao Jing, Liu Jiapeng and Wang Zhao prepared figures 1-6.All authors reviewed the manuscript.
AVAILABILITY OF DATA AND MATERIALS
All relevant data generated or analysed during this study are included in this published article. Other data are not applicable.
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