Introduction

Salinity and drought have always been important abiotic stress factors that restrict agricultural production1,2. Currently, there is approximately 955 million hm2 of saline land in the world. China contains 10% of this land, which is primarily distributed in the northwestern inland and eastern coastal areas. The area of arid and semiarid arable land accounts for approximately one-half of China's national land area, and there is a trend of increasing expansion3,4. Studying the effects of salt damage and drought adversity stress on the growth of pasture grasses and exploring the corresponding regulatory techniques has been a research hotspot on the resistance of pasture grass to stress5,6.

Elymus nutans Griseb. (E. nutans) is a perennial herbaceous plant of the Gramineae family, which is resistant to cold and drought, tolerant to salinity, and characterized by its strong tillering ability and abundant fibrous roots7,8. As an excellent pasture grass in the Qinghai-Tibetan Plateau region in China, it has strong resistance and tolerance to cold, rich nutrients, good palatability, high yield, and tolerance to desert areas among others. It can also prevent winds and fix sands, conserve soil and water, and accelerate the process of ecological restoration of the grassland, which can effectively reduce the economic losses caused by natural disasters9,10. Research suggests that during the initial germination phase, E. nutans seeds exhibit higher tolerance to salt stress than drought stress under the same osmotic potential treatment. The examination of C:N and N:P ratios in E. nutans seedlings indicates that salt stress significantly affects nitrogen utilization and tissue function. Therefore, studying the physiological and hormonal responses of E. nutans seedlings to drought and salt stress is essential for improving our understanding of the drought and salt tolerance mechanisms of E. nutans11.

A large number of studies have shown that changes in the physiological indicators, such as the contents of malondialdehyde (MDA), soluble protein (SP), and proline (Pro) and the activities of peroxidase (POD) and superoxide dismutase (SOD), in plant shoots and roots are related to plant stress tolerance12,13. Auxin was the earliest hormone discovered and regulates shoots morphogenesis14. Cytokinin (CTK) regulates the processes of plant cell division, growth and development and the uptake of nutrients by tissues, and organs, as well as those of individuals and biotic and abiotic stresses among others. Gibberellins (GAs) are a group of essential phytohormones during plant growth and development. They regulate processes, such as seed germination, stem elongation, flower formation, and flower and fruit development15. Abscisic acid (ABA) plays an important role in the regulation of plant cell division and amplification. for example of abscisic acid, somatic embryos could sprout adventitious shoots16,17. In summary, phytohormones both independently and synergistically regulate plant growth and development and the processes of environmental responses. Soil drought and salinity, as two major abiotic stressors that affect plant growth and crop yield, are in some sense both stresses (osmotic stress) imposed by the environmental water potential on plants, and they are both similar to each other and different from each other18. To study the physiological responses of the E. nutans seedling shoots and roots to drought stress and salt stress and the changes of endogenous hormones in E. nutans seedlings, we conducted a pot experiment in an artificial climate incubator in the Grass Laboratory of the Tibet College of Agriculture and Livestock, Linzhi, Tibet.

Materials and methods

Introduction to materials

The experiment was completed in the Grass Science Laboratory of the Tibet College of Agriculture and Animal Husbandry, Tibet Autonomous Region (94°20′31.4″ N, 29°39′57.5″ E). The test material was Elymus nutans Griseb. cv. ‘Baqing’, which was provided by the Grass Science Laboratory of the Tibet College of Agriculture and Animal Husbandry. The experiment was conducted on April 1, 2023, and ended on April 28, for a total of 28 days.

Experimental design

Solutions of PEG-6000 (to simulate drought stress) and sodium chloride (NaCl) (to simulate salt stress) were configured with water potentials of 0 (CK), − 0.04, − 0.14, − 0.29, − 0.49, − 0.73, and − 1.02 MPa19,20. Sand was used as the culture medium, and each germination box (19 × 13 × 12 cm) was lined with 1,000 g of dry sand. A volume of 100 mL of solutions of PEG-6000 and NaCl of different gradients were added, and the seeds were sown at a depth of 2–3 cm with a seed volume of 5 g m−2 after they had been mixed well. To ensure that the environmental water potential was maintained at a constant level, the germination box was placed in a constant temperature incubator at 25 °C using the weighing method, and the distilled water was replenished three times a day – in the morning, in the middle, and in the evening – according to the amount of evaporation in the germination box to ensure that the content of the solution in the sand was around 10%. The level of light in the incubator was 1,250 lx; the photoperiod was 12 h, and the dark period was 12 h. Hoagland's total nutrient solution was replenished every 7 days with 20 mL each time, and the physiological indices of the shoots and roots and the endogenous hormone contents of the seedlings were measured after 28 days. Each treatment involved using 3 pots of materials with consistent growth as 3 biological replicates.

Fresh and dry weight measurement and chlorophyll content determination

After 28 days, 10 seedlings were randomly selected from each treatment germination box. The fresh and dry weights of the shoots and roots of these seedlings were determined. To measure the fresh weight, the shoots and roots were carefully separated and immediately weighed using an analytical balance. For dry weight, the separated shoots and roots were placed in a drying oven at 70 °C for 48 h until a constant weight was achieved and then weighed again.

The relative water content (RWC) was calculated using the formula:

$${\text{RWC}}\left({\%}\right)=\frac{\left({\text{FW}}-{\text{DW}}\right)}{\left({\text{TW}}-{\text{DW}}\right)}\times 100$$

where FW is the fresh weight, DW is the dry weight, and TW is the turgid weight (measured after soaking the plant material in distilled water for 24 h).

Chlorophyll a and b contents were determined by ethanol extraction. Specifically, 0.1 g of fresh leaf tissue was homogenized in 10 mL of 95% ethanol and kept in the dark for 24 h. The extract was then centrifuged at 3000 rpm for 10 min. The supernatant was collected and its absorbance was measured at 665 nm for chlorophyll a and 649 nm for chlorophyll b using a spectrophotometer21.

Enzyme activity and biochemical analysis

The activity of superoxide dismutase (SOD) was determined by the nitroblue tetrazolium (NBT) photoreduction method. Leaf samples (0.5 g) were homogenized in 5 mL of 50 mM phosphate buffer (pH 7.8) and the homogenate was centrifuged at 12,000 rpm for 20 min. The reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, and 100 µL enzyme extract. The mixture was incubated under light for 15 min and the absorbance was measured at 560 nm21.

Peroxidase (POD) activity was determined using the guaiacol method. Briefly, 0.5 g of leaf tissue was homogenized in 5 mL of 50 mM phosphate buffer (pH 7.0). The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 20 mM guaiacol, 10 mM H₂O₂, and 100 µL of enzyme extract. The increase in absorbance was measured at 470 nm for 1 min22.

Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) colorimetric method. Leaf tissue (0.5 g) was homogenized in 5 mL of 5% trichloroacetic acid (TCA) and centrifuged at 10,000 rpm for 20 min. The supernatant was mixed with an equal volume of 0.6% TBA in 10% TCA and heated at 95 °C for 30 min, then cooled quickly on ice. After centrifugation at 10,000 rpm for 10 min, the absorbance of the supernatant was measured at 532 nm and 600 nm22.

Proline (Pro) content was determined by the ninhydrin colorimetric method. Leaf samples (0.5 g) were homogenized in 10 mL of 3% sulfosalicylic acid and filtered. The reaction mixture composed of 2 mL filtrate, 2 mL glacial acetic acid, and 2 mL ninhydrin reagent was heated at 100 °C for 1 h. The reaction was terminated in an ice bath, and the mixture was extracted with 4 mL toluene. The chromophore containing toluene was measured at 520 nm22.

Soluble protein (SP) content was determined using Coomassie Brilliant Blue G-250. A sample homogenate was prepared as described in the Bradford method, and the absorbance was measured at 595 nm22,23.

Hormone analysis

The content of endogenous hormones was determined by ultra-high performance liquid chromatography (UPLC) using a Waters Acquity system (Waters, Milford, MA, USA) and mass spectrometry SCIEX AB 5500 QQQ-MS (SCIEX AB, Redwood City, CA, USA). Hormone extraction was performed on 0.1 g of fresh leaf tissue, which was homogenized in 1 mL of 80% methanol containing 0.1% formic acid, followed by centrifugation at 12 000 rpm for 15 min at 4 °C. The supernatant was filtered through a 0.22 µm membrane and used for UPLC-MS analysis.

A volume of 0.1 mL of standard solution (10.0 µg mL⁻1) was removed, and the standard solution was diluted to 1.0 mL with 80% aqueous methanol to form a standard solution of 1,000 ng mL⁻1. Serial dilutions were prepared with 80% aqueous methanol to obtain appropriate standard concentrations for calibration curves.

The hormone indicators included gibberellins (GA1, GA3, GA4, and GA7), 1-naphthylacetic acid (NAA), indole-3-acetic acid (IAA), 3-indolepropionic acid (IPA), 3-indolebutyric acid (IBA), 2-ip-riboside, kinetin (KT), 6-benzylaminopurine (6-BA), 6-(y,y-dimethylallylaminopurine) (2-IP), trans-zeatin (TZ), trans-zeatin riboside (TZR), DL-dihydrozeatin (DHZ), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and melatonin (MT), which were quantified using multiple reaction monitoring mode.

Statistical analysis

MultiQuant software (SCIEX) was utilized for integration, and a standard curve was used to calculate the contents of hormones24. For the statistical analysis of physiological indices and endogenous hormone contents in the shoots and roots of seedlings under PEG-6000 or NaCl stress, a one-way analysis of variance (ANOVA) was performed using SPSS software (SPSS Inc., Chicago, IL, USA). The ANOVA was used to test the significance of the treatment effects, and post-hoc comparisons were conducted using Tukey’s test at a significance level of P < 0.05 and P < 0.0125. Origin Pro 2021 (OriginLab, Northampton, MA, USA) was used to analyze the principal component analysis of the environmental water potential and physiological indicators of PEG-6000 and NaCl26. A Mantel test was used to assess the correlation between the matrix of endogenous hormone levels and the matrix of physiological indices in E. nutans seedlings. This test was performed to evaluate the strength and significance of associations between hormone levels and physiological responses. Structural equation modeling (SEM) was employed using SPSS AMOS (IBM, Armonk, NY, USA) to explore the relationships among endogenous hormones, and the accumulation of water and dry matter in the shoots and roots of seedlings. The SEM analysis allowed us to model complex relationships and infer direct and indirect effects between variables27. To compare the drought tolerance and salt tolerance of E. nutans seedlings, the subordinate function method was used. The formula for calculating the drought or salt tolerance index was as follows:

$$X(u)=\frac{X-{X}_{{\min}}}{{X}_{{\max}}-{X}_{{\min}}}$$

where X represents the observed value, Xmin represents the minimum value observed, and.

Xmax represents the maximum value observed. For indices that were negatively correlated with drought or salt tolerance, the formula was adjusted as:

$$X(u)=1-\frac{X-{X}_{{\min}}}{{X}_{{\max}}-{X}_{{\min}}}$$

The coefficient of variation (CV) of hormone contents was calculated using SPSS to assess the variability in hormone levels under different treatments. The CV was expressed as a percentage and calculated using the formula:

$$CV\left({\%}\right)=\frac{{\text{Standard\, Deviation}}}{{\text{Mean}}}\times 100$$

Results

Effect of treatments on the accumulation of biomass in E. nutans seedlings

The accumulation of shoot biomass indicated a significant reduction in the fresh weight of E. nutans shoots under simulated drought stress with partial treatment of PEG-6000. The fresh weight was notably lower compared to the control (CK) at − 0.14 MPa, showing a 17% decrease to 129.67 mg·10 plant−1 (Fig. 1A). Similarly, NaCl stress led to a significant decrease in fresh weight, with the lowest value observed at 148.67 mg·10 plant−1 under − 1.02 MPa treatment (P < 0.01) (Fig. 1A).The reduction in ambient water potential also impacted the accumulation of dry matter by the shoots. Dry weight under PEG-6000 treatment at − 0.14 MPa significantly decreased by around 12% compared to the control, reaching its lowest value of 52.33 mg·10 plant−1 under − 1.02 MPa treatment (Fig. 1B). Under NaCl stress, dry weight was significantly lower than the CK, with the lowest value observed at 55.67 mg·10 plant−1 under − 1.02 MPa treatment (Fig. 1B).Relative water content exhibited varying trends under both PEG-6000 and NaCl stress conditions. Under PEG-6000 stress, the relative water content increased initially and then decreased, while under NaCl stress, it was slightly higher than the control under certain treatments but significantly lower at − 0.29 MPa, reaching a minimum of 62.55% under − 1.02 MPa treatment (Fig. 1C).

Figure 1
figure 1

Effects of two solutions on biomass accumulation. Two types of solution stress affect E. nutans Shoots fresh weight (A), shoots dry weight (B), Relative shoots water content (C), Root fresh weight (D), root dry weight (E), and Relative water content of the root system (F). Lowercase letters denote significant differences in the same solution under different treatments (P < 0.05), while uppercase letters signify extremely significant differences in the same solution under different treatments (P < 0.01).

Full size image

In terms of root biomass accumulation, the fresh weight of E. nutans roots initially increased and then decreased with decreasing ambient water potential. Both fresh weight and dry weight of the roots were significantly higher than those of the control (CK) at − 0.04 MPa treatment (P < 0.05) (Fig. 1D, E). However, as the ambient water potential decreased, both the fresh weight and dry weight of the root system decreased to their lowest values at − 1.02 MPa treatment, measuring 113.33 and 19.67 mg·10 plant−1, respectively (Fig. 1D, E). Additionally, the relative water content of the E. nutans root system decreased in alignment with the ambient water potential, dropping to approximately 82.62% at − 1.02 MPa treatment (Fig. 1F).Under NaCl stress, the fresh weight and dry weight of the root system showed a similar trend of initial increase followed by a decrease with decreasing ambient water potential. The maximum values were significantly higher than the control (CK) at − 0.14 MPa treatment (P < 0.05). Subsequently, as the ambient water potential decreased further, both the fresh weight and dry weight of the root system declined to their lowest values at − 1.02 MPa treatment, measuring 75.67 and 11.33 mg·10 plant−1, respectively (Fig. 1D, E). Likewise, the relative water content of the E. nutans root system under NaCl stress exhibited a similar increasing-then-decreasing trend, reaching its lowest value of 85.02% at − 1.02 MPa treatment (Fig. 1F).

Effect of treatments on the shoots physiology of E. nutans seedlings

In terms of chlorophyll, the simulation of drought stress by PEG-6000 extremely significantly reduced the contents of chlorophyll a and chlorophyll b in the E. nutans shoots. These values were extremely significantly lower than those of the CK in all the treatments (P < 0.01), with a minimum in the − 1.02 MPa treatment with the contents of chlorophyll a and chlorophyll b of 2.34 and 2.13 mg g−1, respectively (Fig. 2A, B). Under salt stress, the contents of chlorophyll a and chlorophyll b in E. nutans decreased as the ambient water potential of the NaCl treatment decreased. When the ambient water potential reached − 0.04 MPa, the content of chlorophyll a was extremely significantly lower than that of the CK (P < 0.01). However, the content of chlorophyll b was only extremely significantly lower than that of the CK (P < 0.01) in the − 0.14 MPa treatment. The contents of both chlorophyll a and chlorophyll b were lower in the − 1.02 MPa treatment. The content of chlorophyll b was the lowest from both stress treatments in the − 1.02 MPa treatment at 3.01 and 3.59 mg g−1, respectively (Fig. 2A, B). Under PEG-6000 stress, the activities of both POD and SOD in the shoots increased and then decreased with the decrease in ambient water potential, and the activities of the two enzymes reached their maximum value in the − 0.73 MPa treatment and were 71.88 g min−1 FW and 323 U·(g FW−1), respectively (Fig. 2C, D). Under NaCl stress, the activities of both POD and SOD also decreased with the decrease in the ambient water potential. Both showed a similar trend of increasing and then decreasing with the decrease in ambient water potential, and the activities of both enzymes reached a maximum value of 76.10 g min−1 FW and 361 U·(g FW−1) in the − 0.73 MPa treatment (Fig. 2C, D). In terms of shoots plasma membrane oxidation products and cellular osmoregulatory substances, the MDA content of E. nutans shoots under PEG-6000 and NaCl stress increased with the decrease in ambient water potential with the highest contents of 32.44 and 32.43 m mol g−1 FW in the − 1.02 MPa treatment under both types of stresses, respectively (Fig. 2E). The SP content also showed the same trend (increasing and then decreasing) and also reached maximum values of 81.02 and 96.18 mg g−1 FW in the − 0.73 MPa treatment, respectively (Fig. 2F). The Pro content also showed the same trend (decreasing and then increasing) under both stresses and reached maximum values of 13.2 and 15.73 μg g−1 FW in the − 1.02 MPa treatment, respectively (Fig. 2G).

Figure 2
figure 2

Effects of two solutions on Shoots Physiological Indicators. Two types of solution stress affect E. nutans Chlorophyll a content (A),Chlorophyll b content (B), shoots POD activated (C), shoots SOD activated (D), shoots Pro content (E), shoots SP content (F), and shoots MDA content (G). Lowercase letters denote significant differences in the same solution under different treatments (P < 0.05), while uppercase letters signify extremely significant differences in the same solution under different treatments (P < 0.01).

Full size image

Effect of treatments on the root physiology of E. nutans seedlings

The activities of enzymes in the roots in response to the PEG-6000 and NaCl stress were basically the same. The activities of POD also increased with the reduction in ambient water potential and reached a maximum value of 26.50 and 27.16 g min−1 FW in the − 1.02 MPa treatment, respectively (Fig. 3A). The activities of SOD were basically the same as those of the POD. The activity increased and decreased with the reduction in ambient water potential, and the activity of the enzyme in both treatments also reached a maximum value of 289 and 298 U·(g FW−1) in the − 0.73 MPa treatment, respectively (Fig. 3B). The activities of SOD and POD were basically the same with the decrease in ambient water potential. They increased and then decreased, and the two treatments also reached the maximum values of 289 and 298 U·(g FW−1) in the − 0.73 MPa treatment, respectively (Fig. 3B). The effects of PEG-6000 and NaCl stress on the root system of E. nutans seedlings in terms of plasma membrane oxidation products and cellular osmotic regulatory substances were basically the same. With the decrease in ambient water potential, the content of MDA in the root system increased and reached its maximum values of 35.04 and 33.07 m mol g−1 FW in the − 1.02 MPa treatment, respectively (Fig. 3D). The content of SP increased and then decreased and reached maximum values of 58.32 and 66.66 mg g−1 FW in the − 0.73 MPa treatment (Fig. 3C). The content of Pro increased and reached maximum values of 58.32 and 66.66 mg g−1 FW from the − 1.02 MPa treatment and reached maximum values of 40.74 and 54.45 μg g−1 FW, respectively (Fig. 3E).

Figure 3
figure 3

Effects of two solutions on Shoots Physiological Indicators. Two types of solution stress affect E. nutans root POD activated (A), root SOD activated (B), root SP content (C), root MDA content (D) and shoots Pro content (E). Lowercase letters denote significant differences in the same solution under different treatments (P < 0.05), while uppercase letters signify extremely significant differences in the same solution under different treatments (P < 0.01).

Full size image

Effect of treatments on the content of GA in E. nutans seedling shoots

Under the drought stress simulated by PEG-6000, the content of GA1 in E. nutans shoots decreased and then increased as the ambient water potential decreased. It reached a minimum of 0.22 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01) and reached a maximum of 5.55 pg mg−1 in the − 1.02 MPa treatment, which was significantly higher than that of the CK (P < 0.01) (Fig. 4A). The content of GA3 was significantly lower than that of the CK (P < 0.05) in the − 0.04 MPa treatment. It gradually increased with the decrease in ambient water potential and reached a maximum value of 2.86 pg mg−1 in the − 0.29 MPa treatment before decreasing with the further decrease in the ambient water potential (Fig. 4B). The content of GA4 decreased with the decrease in ambient water potential (Fig. 4B). The content decreased and then increased with the decrease in the ambient water potential and reached a minimum of 2.11 pg mg−1 in the − 0.04 MPa treatment, which was highly significant lower than that of the CK (P < 0.01). It reached a maximum value of 5.56 pg mg−1 in the − 1.02 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 4C). The content of GA7 showed the same trend as that of GA3, which also occurred in the − 0.29 MPa treatment. This value was highly significantly higher than that of the CK (P < 0.01) and also reached a maximum value of 4.23 pg mg−1 in the − 0.29 MPa treatment (Fig. 4D).

Figure 4
figure 4

Effects of two solutions on GA content. Two types of solution stress affect E. nutans GA1 content (A), GA3 content (B), GA4 content (C) and GA7 content (D). Lowercase letters denote significant differences in the same solution under different treatments (P < 0.05), while uppercase letters signify extremely significant differences in the same solution under different treatments (P < 0.01).

Full size image

Under the salt stress simulated by NaCl, the content of GA1 in E. nutans also showed the same trend of decreasing and then increasing with the decrease in ambient water potential. It reached a minimum of 0.51 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01) and a maximum of 5.35 pg mg−1 in the − 1.02 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 4A). The content of GA3 was significantly lower than that of the CK in the − 0.04 MPa treatment (P < 0.05) and gradually increased with the decrease in the content of GA3 in the ambient water potential. It reached a maximum value of 1.89 pg mg−1 in the − 0.29 MPa treatment and decreased with the further decrease in the ambient water potential; the treatments under the stress of NaCl GA3 content was lower than that of the CK in all the treatments under NaCl stress (Fig. 4B). The GA4 content decreased and then increased with the decrease in the ambient water potential. It reached a minimum of 3.43 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01) and reached a maximum value of 5.18 pg mg−1 in the − 1.02 MPa treatment, which was significantly higher than that of the CK (P < 0.05) (Fig. 4C). The content of GA7 followed the same trend as that of the GA3 with a decreasing-increasing–decreasing trend. It reached a minimum of 3.22 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01). It reached its maximum value of 3.89 pg mg−1 in the − 0.73 MPa treatment, which was not statistically significant (Fig. 4D).

Effect of treatments on the contents of growth hormones in the E. nutans seedlings shoots

Under the drought stress simulated by PEG-6000, the content of IAA in the E. nutans shoots increased and then decreased with the decrease in ambient water potential. It reached a maximum value of 105.06 pg mg−1 in the − 0.73 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 5A). The content of IPA increased and then decreased with the decrease in ambient water potential. It reached a maximum value of 169.65 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 5A). The content of IPA in the 0.04 MPa treatment reached a maximum value of 169.65 pg mg−1, which was highly significantly higher than that of the CK (P < 0.01). It reached a minimum value of 41.18 pg mg−1 in the − 1.02 MPa treatment, which was highly significantly lower than that of the CK (Fig. 5B). The content of IBA decreased as the ambient water potential decreased, which was highly significantly lower than that of the CK (P < 0.01) in all the treatments. The content of riboside showed a decreasing-increasing–decreasing trend with the decrease in the ambient water potential. It reached a minimum of 4.02 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01) and a minimum of 5.5 pg mg−1 in the − 0.73 MPa treatment (Fig. 5B). The content of NAA decreased with the decrease in ambient water potential, which was significantly lower than that of the CK in all the treatments (P < 0.05). The lowest value was in the − 1.02 MPa treatment, which was approximately 78.5% lower than that of the CK (Fig. 5E).

Figure 5
figure 5

Effects of two solutions on auxin content. Two types of solution stress affect E. nutans IAA content (A), IPA content (B), IBA content (C) and 2ip-riboside content (D) and NAA content (E). Lowercase letters denote significant differences in the same solution under different treatments (P < 0.05), while uppercase letters signify extremely significant differences in the same solution under different treatments (P < 0.01).

Full size image

Under the salt stress simulated by NaCl, the IAA content in the shoots of common reed (Phragmites australis) increased and then decreased with the decrease in the ambient water potential and reached a maximum value of 37.74 pg mg−1 in the − 0.29 MPa treatment, which was significantly higher than that of the CK (P < 0.01) (Fig. 5A). The content of IPA decreased with the decrease in ambient water potential, which was significantly lower than that of the CK in all the treatments (P < 0.05). It reached a minimum value of 73.24 pg mg−1 in the − 1.02 MPa treatment, which was highly significantly lower than that of the CK (Fig. 5B). The content of IBA decreased in parallel with the ambient water potential and was highly significantly lower than the CK in all the treatments (P < 0.01); the lowest value was observed in the − 1.02 MPa treatment where the content decreased by approximately 56.3% (Fig. 5C). The content of 2 ip-riboside decreased and then increased as the water potential decreased and reached a minimum of 3.4 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01). It reached a maximum of 5.54 pg mg−1 in the − 1.02 MPa treatment, which did not reach a significant level of difference with the CK (Fig. 5D). The content of NAA decreased with the decrease in the ambient water potential, which was highly significantly lower than that of the CK in all the treatments except for the treatment of − 0.04 MPa (P < 0.01). It reached its minimum value in the − 1.02 MPa treatment, which was approximately 49.7% lower than that of the CK (Fig. 5E).

Effect of treatments on the contents of cytokinins in the E. nutans seedlings shoots

Under the drought stress simulated by PEG-6000, the content of 6-BA in the E. nutans shoots increased and then decreased with the decrease in ambient water potential, and the highest content was 1.38 pg mg−1 in the − 0.73 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 6A). The content of 2-IP increased with the decrease in ambient water potential, and the highest content was 2.96 pg mg−1 in the − 1.02 MPa treatment (Fig. 6B). The content of KT increased and then decreased with the highest content of 1.84 pg mg−1 in the − 0.73 MPa treatment, which was highly significantly higher than that of the CK and other treatments (P < 0.01) (Fig. 6C). The content of DHZ showed a decreasing-increasing–decreasing trend with the decrease in ambient water potential. The lowest amount was 3.9 pg mg−1 in the − 0.04 MPa treatment, which did not reach the level of a significant difference with that of the CK; the highest content was 7.07 pg mg−1 in the − 0.49 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 6D). The content of TZ increased and then decreased with the decrease in ambient water potential and the highest content of 3.9 pg mg−1 in the − 0.73 MPa treatment, which was highly significantly higher than that of the CK and the other treatments (P < 0.01) (Fig. 6E). The content of TZR showed a decreasing-increasing–decreasing trend with the decrease in ambient water potential. The content was the lowest at 12.75 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01). The content was the highest at 23.75 pg mg−1 in the − 0.73 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01). (Fig. 6F).

Figure 6
figure 6

Effects of two solutions on cytokinin content. Two types of solution stress affect E. nutans 6-BA content (A), 2.IP content (B), KT content (C), DHZ content (D), TZ content (E) and TZR content (F). Lowercase letters denote significant differences in the same solution under different treatments (P < 0.05), while uppercase letters signify extremely significant differences in the same solution under different treatments (P < 0.01).

Full size image

Under the salt stress simulated by NaCl, the content of 6-BA in the E. nutans shoots increased and then decreased with the decrease in ambient water potential, with the highest content of 0.63 pg mg−1 in the − 0.29 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 6A). The content of 2-IP increased and then decreased with the decrease in ambient water potential, with the highest content of 1.43 pg mg−1 in the − 0.29 MPa treatment. The highest content was 1.43 pg mg−1 in the − 0.29 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 6B). The content of KT increased and then decreased, and the highest content was 0.74 pg mg−1 in the treatment − 0.29 MPa, which was highly significantly higher than that of the CK and the other treatments (P < 0.01) (Fig. 6C). The content of DHZ showed a decreasing-increasing–decreasing trend with the decrease in environmental water potential and was highly significantly higher than that of the CK and the other treatments (P < 0.01) (Fig. 6C). The content of DHZ showed a decreasing-increasing–decreasing trend with the decrease in the ambient water potential, with the highest content of 5.09 pg mg−1 in the − 0.29 MPa treatment, which did not reach the level of a significant difference with the CK (Fig. 6D). The content of TZ increased with the decrease in ambient water potential, with the highest content of 38.35 pg mg−1 in the − 1.02 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01) (Fig. 6E). The content of TZR decreased and then increased with the decrease in ambient water potential. The lowest content was 6.6 pg mg−1 in the − 0.04 MPa treatment, which was highly significantly lower than that of the CK (P < 0.01). The highest content was 25.56 pg mg−1 in the − 1.02 MPa treatment, which was highly significantly higher than that of the CK (P < 0.01)(Fig. 6F).

Effect of treatments on the content of abscisic acid and other endogenous substances in the shoots of E. nutans seedlings

Under the drought stress simulated by PEG-6000, the content of ABA in the E. nutans shoots increased with the decrease in ambient water potential, and the highest content was 46.58 pg mg−1 in the − 1.02 MPa treatment, which was highly significantly higher than that in the CK (P < 0.01) (Fig. 7A). The content of SA decreased with the decrease in ambient water potential, and the lowest content was observed in the − 1.02 MPa treatment, which was highly significantly lower than that in the CK by approximately 78.25% (Fig. 7B). The content of JA decreased with the decrease in ambient water potential, and the lowest content was found in the − 1.02 MPa treatment, with a decrease of approximately 59.23% (Fig. 7C). The content of MT increased and then decreased with the decrease in ambient water potential, with the highest content of 8.5 pg mg−1 in the − 0.04 MPa treatment; the highest content was found to be 8.5 pg mg−1, which was highly significantly (P < 0.01) higher than that of the CK (Fig. 7D).

Figure 7
figure 7

Effects of two solutions on Abscisic acid and other substances content. Two types of solution stress affect E. nutans ABA content (A), SA content (B), JA content (C) and MT content (D). Lowercase letters denote significant differences in the same solution under different treatments (P < 0.05), while uppercase letters signify extremely significant differences in the same solution under different treatments (P < 0.01).

Full size image

Comprehensive analysis

The fresh weight, dry weight and relative water content of the E. nutans shoots and roots after the simulation of drought stress using PEG-6000 positively correlated with the contents of chlorophyll a and chlorophyll b and the environmental water potential and negatively correlated with the activities of POD and SOD and the contents of MDA, Pro, and SP in the shoots and roots (Fig. 8A). The accumulation of water and dry matter in the shoots and roots highly significantly correlated (P < 0.01) with the endogenous hormones measured; the physiological indices of shoots and roots, which did not significantly correlate with GA3, also highly significantly correlated (P < 0.05) with the other endogenous hormones measured (Fig. 8C). The growth hormones, ABA and other endogenous substances inhibited the production of cytokinins, while the GAs enhanced their production; the cytokinins enhanced the production of GAs, ABA and other endogenous substances, and the endogenous hormones did not reach the level of a significant difference in the structural equation modeling of the accumulation of water and dry matter in both the shoots and roots (Fig. 8E).

Figure 8
figure 8

Comprehensive analysis. Principal component analysis of PEG-6000 on Physiological indicators (A); principal component analysis of NaCl on Physiological indicators (B); Comprehensive analysis. Mantel test analysis of PEG-6000 on Physiological indicators and Hormonal indicators (C); Mantel test analysis of NaCl on Physiological indicators and Hormonal indicators (D); Comprehensive analysis. Structural equation modeling (SEM) analysis of PEG-6000 on Interaction between endogenous hormones and shoots and root biomass accumulation (E); Structural equation modeling (SEM) analysis of NaCl on Interaction between endogenous hormones and shoots and root biomass accumulation (F). (LFW, shoots fresh weight; LDW, shoots dry weight; RFW, roots fresh weight; RDW, roots dry weight; LW, shoots moisture content; RW,roots moisture content; L-SOD, shoots SOD; R-SOD,roots SOD; L-POD, shoots POD; R-POD, roots POD; L-Pro, shoots Pro; R-Pro, roots Pro; L-MDA, shoots MDA; R-MDA, roots MDA; L-SP, shoots SP; R-SP, roots SP. Table 1 same).

Full size image

The fresh weight, dry weight, and relative water content of the E. nutans shoots and roots after the simulation of salt stress by NaCl positively correlated with the contents of chlorophyll a and chlorophyll b and the ambient water potential; and negatively correlated with the activities of POD and SOD and contents of MDA, Pro, and SP in the shoots and roots (Fig. 8B). The accumulation of shoots biomass did not significantly correlate with the contents of GA7, IAA, 2.IP, KT, 6-BA, DHZ, and JA; the accumulation of root biomass did not significantly correlate with the contents of GA7, IAA, 2.IP, KT, 6-BA, DHZ, and MT; the shoots physiological indices did not significantly correlated with IAA, 2.IP, 6-BA, and DHZ; and the root physiological indices that did not reach significant correlation with IAA, KT, and DHZ (Fig. 8D). The growth hormones and ABA and other endogenous substances enhanced the production of cytokinins, while the GAs inhibited the production of these hormones; cytokinins, growth hormones, ABA, and other endogenous substances inhibited the production of GAs; GAs and the growth hormones inhibited the production of ABA and other endogenous substances; and cytokinins inhibited the accumulation of water and dry matter by the shoots (Fig. 8F).

Discussion

Effects of drought stress and salt stress on the biomass accumulation of E. nutans seedlings

The seedling stage is a very sensitive stage of plant growth and development to water, and water stress in the seedling stage will not only affect plant growth and development, which leads to a decrease in yield, but can also cause plant death in severe cases. The final result caused by the water deficit will be synthesized in the biomass of shoots and roots, which is a positive indicator to evaluate the resistance of forage grasses to drought and salinity. In this study, we found that the fresh and dry weight of the E. nutans shoots under the same environmental water potential decreased with the decrease in environmental water potential under drought stress and salt stress, which was similar to the results of Ahmed et al.28 and Calon et al.29. In addition, we found that the fresh and dry weight of the E. nutans shoots under NaCl stress were higher than those under the same environmental water potential. Treatment with PEG-6000 caused stress at the water potential, which could be owing to the stronger inhibition of E. nutans shoots growth by drought stress than salt stress.

In the absence of water, plants will compensate for the reduction in the area of root area by enhancing root vigor and maintaining a higher root biomass to maintain a high ability of the root to absorb water. In this study, we found that the two water deficit stressors resulted in "low promotion and high suppression" of the fresh and dry weights of E. nutans roots, which was similar to the results of the studies conducted by Chang et al.30 and Pang et al.31. The E. nutans roots under drought stress had higher fresh weights than those under salt stress when the ambient water potential was less than − 0.14 MPa, and the dry weight of E. nutans roots under all the PEG-6000 treatments was higher than that of the NaCl treatment, which could be owing to the decrease in ambient water potential. This could be owing to the stress of drought and salt damage to the root system and the stronger inhibitory effects of Na+ and Cl− on plant root growth than a single drought stress.

Effects of drought stress and salt stress on the physiological indices of E. nutans seedlings

The study of physiological responses of plants to drought stress and salt stress helps to reveal the physiological mechanisms of plant adaptation to water adversity. During photosynthesis, with the water deficit and accumulation of reactive oxygen species (ROS), the synthesis of chlorophyll in the chloroplasts is blocked, while its degradation is promoted, which results in a decrease in the content of chlorophyll in plants. In this study, we found that the 2 water deficits resulted in the reduction of the contents of chlorophyll a and chlorophyll b in the shoots, which was similar to the results of Tsai et al.32 and Bano et al.33. However, the chlorophyll a content of E. nutans was higher than that of the PEG-6000 treatment in all the treatments except the − 0.04 MPa treatment, which was lower than that of PEG-6000, which could indicate that the photosynthetic capacity of E. nutans in the salinized environment was stronger than that of drought stress under the same environmental water potential.

Studies have shown that under some degree of drought stress, plants are not damaged by ROS, which is owing to the fact that the antioxidant enzyme system in the plant tissues will scavenge the ROS generated by the stress, so that their amount can be controlled in a safe range. POD and SOD play important roles during the process of drought stress and salt stress in the plant, and the amount of their activity has some correlation with the resistance of plants to drought and salinity. Higher activity results in enhanced resistance to drought and salt in plants. In this study, we found that the antioxidant enzyme systems (POD and SOD) of the shoots and roots of the E. nutans seedlings under the two stress treatments increased and then decreased with the reduction in ambient water potential, which is similar to the results of Yildiztekin et al.34 and Jiao et al.35. Moreover, in this study, the salt stress of E. nutans seedlings under the same ambient water potential was found to increase and then decrease, which is similar to the findings of Yildiztekin et al.34 and Jiao et al.35. All the treatments of E. nutans seedling shoots and root antioxidant enzyme systems (POD and SOD) were higher when subjected to salt stress than drought stress, which could indicate that the inhibitory effect of salt stress on plant shoots and roots is primarily reflected in the growth of roots, and the effect of salt stress on the antioxidant enzyme systems of plants is weaker than that of drought stress.

Changes in the content of MDA under drought stress can cause damage that reflects the degree of lipid peroxidation in plant membranes. A higher content of MDA results in more serious damage to the plant cell membranes. In this study, the content of MDA in the shoots and roots of E. nutans increased with the decrease in ambient water potential, which was similar to the findings of Gulzar et al.36 and Zhao et al.37. In addition, the content of MDA in the shoots and roots of E. nutans under the same ambient water potential under the PEG-6000 treatment was found to cause more stress than the NaCl treatment. The accumulation of MDA in the shoots and roots of E. nutans under PEG-6000 at the same environmental water potential was higher than that under NaCl, which could be owing to the fact that the membrane lipid peroxidation reaction of E. nutans under PEG-6000 stress was stronger than that under NaCl stress, which resulted in more serious damage to the cell membranes.

The high or low contents of SP and Pro can reflect the magnitude of drought and salt tolerance in plants, and both of them, as the primary osmoregulatory substances in cells, can alleviate or resist the occurrence of damage under the difficulty of stress. We found that the shoots and roots of E. nutans reacted differently in response to environmental water potential stress. The content of Pro in the shoots decreased and then increased with the reduction in environmental water potential, while the content of Pro in the roots increased with the reduction in environmental water potential. The content of SP in the shoots and roots increased and then decreased with the reduction in environmental stress, which was consistent with the results of Kim et al.38 and FarMohammadi et al.39. Moreover, this study found that the contents of SP and Pro in the shoots and roots of E. nutans treated with NaCl were higher than those of PEG-6000 under the same environmental water potential, which could be owing to the higher osmotic pressure imbalance of the E. nutans seedlings owing to salt stress compared with drought stress, and the content of soluble proteins was reduced.

The effects of PEG-6000 and NaCl on the physiology of E. nutans seedlings at the same ambient water potential showed different results, and the analysis of the physiological indices by the affiliation function method revealed that E. nutans was better able to tolerate salt than drought at the same ambient water potential (Table 1).

Table 1 Table of affiliation functions of physiological indicators.
Full size table

Effects of drought stress and salt stress on endogenous hormones and other substances in E. nutans seedlings

GAs, as a group of the most important phytohormones, control different aspects of plant growth and development, including seed germination, stem elongation and flower induction, and are important hormones for plant regulation under adverse conditions40. In this study, GA1 showed exactly the same trend of decreasing and then increasing under the two stresses of PEG-6000 and NaCl, which was similar to the results of Liu et al.41. However, under lower ambient water potential treatments, the content of GA1 was lower following NaCl stress than that of PEG-6000, which could be owing to the fact that the response of GA1 to NaCl was more intermediate under lower ambient water potential conditions, which led to an increase in the regulation of GA1 as the degree of drought stress intensified. The trend of the regulation of contents of GA4 was the same as that of GA1, but the content of GA4 under NaCl stress was only lower than that of PEG-6000 in the − 1.02 MPa treatment, which could be owing to the late action of the regulatory mechanism of GA4 in the face of drought stress and the more direct action of NaCl stress. GA3 and GA7 showed the same trend of increasing and then decreasing, but under the PEG-6000 stress, the content of GA3 changed dramatically and decreased, while there was no obvious trend in the magnitude of GA3 changes under NaCl stress (Table 2). GA7 also showed a large fluctuating trend under PEG-6000 stress, while the regulation of GA7 content was smaller under NaCl stress. This difference could be owing to the fact that in the face of drought stress, E. nutans seedlings actively regulate both GA3 and GA7 to resist the damage induced by drought, while the regulation of GA3 and GA7 was not very sensitive to the stress of NaCl.

Table 2 Table of coefficients of variation of endogenous hormones.
Full size table

The major natural growth hormone in plants, IAA, orchestrates a large number of developmental responses that are largely dependent on the formation of gradients of growth hormones in plant tissues42. IAA metabolism, including biosynthesis, splicing, and degradation, regulates the gradients of growth hormones along with their intercellular and intracellular transport and therefore, is critical for plant growth43. In this study, we found that the content of IAA in the E. nutans shoots was regulated to a greater extent under drought stress conditions than under salt stress, which could be attributed to the fact that IAA is the major hormone for drought tolerance in E. nutans seedlings and responds relatively weakly to salt stress. The magnitude of regulation of the contents of IPA and IBA under drought stress was similarly greater than that under salt stress, and the contents of IPA and IBA showed relatively stable contents at higher ambient water potentials (− 0.49, − 0.73, and − 1.02 MPa) for the two treatments, which could be owing to the insensitivity of contents of IPA and IBA to the response at low ambient water potentials. The content of 2ip-riboside reacted highly similarly to drought stress and salt stress, and there was only a small magnitude of the effect of the two solutions on the content of 2ip-riboside. This could indicate that 2ip-riboside is not the primary regulatory hormone involved in the response of E. nutans to the ambient water potential. The magnitude of the response to drought stress was significantly larger than that to salt stress, which could perhaps be supplied by means of exogenous NAA during drought tolerance in E. nutans (Table 2).

Plant cytokinins play multiple roles in plant development and affect many agriculturally important processes, including growth, nutrient responses, and responses to biotic and abiotic stresses44. In this study, 6-BA, 2.IP, TZ, KT, and DHZ were found to maintain relatively stable regulatory results under PEG-6000 stress at treatments with higher ambient water potentials (− 0.04, − 0.14, and − 0.29 MPa) except for a higher magnitude of variation at lower ambient water potentials (− 0.49, − 0.73, and − 1.02 MPa) treatments, which could indicate that E. nutans seedlings rely on the regulation of the five hormones 6-BA, 2.IP, TZ, KT, and DHZ to adapt to drought stress under severe drought conditions. Under NaCl stress, 6-BA, 2.IP, TZ, and KT maintained a relatively stable state with a small magnitude of change. Another possibility is that the four hormones 6-BA, 2.IP, TZ, and KT play an important role in the TZR content in E. nutans seedlings that was higher than that of NaCl stress. However, when the ambient water potential reached the degree of NaCl stress induced by the − 1.02 MPa treatment, the content of TZR was higher than that of the PEG-6000 stress. It could also indicate that the accumulation of TZR under drought stress decreased following the drought stress treatment at − 1.02 MPa, whereas it still accumulated in the NaCl stress treatment at − 1.02 MPa. Another possibility is that the accumulation of TZR in the E. nutans seedlings decreased in the treatment at drought stress at − 1.02 MPa but still showed a trend of increasing accumulation in the treatment of PEG-6000, or it could indicate that TZR played an important role in response to salt stress (Table 2).

ABA is an important phytohormone that regulates plant growth, development and stress response. It plays a crucial role in a variety of physiological processes in plants, including stomatal closure, shoots senescence, bud dormancy, seed germination, osmotic regulation, and growth inhibition among others. Similarly, JA, SA, and MT are important endogenous substances that regulate plant growth and improve plant stress tolerance45,46,47. In this study, we found that the content of ABA increased in the same manner under the two stresses, but it increased significantly more in the PEG-6000 stress treatment than in the NaCl stress, which could indicate that ABA plays a greater role in drought resistance than salt resistance in E. nutans seedlings. Alternatively, the trends of SA and JA contents showed completely opposite results to those of ABA. This could indicate that SA and JA play a greater role in salt resistance than drought resistance in E. nutans. The contents of MT basically consistently decreased in response to the decrease in ambient water potentials with the two stresses. However, at lower ambient water potentials (− 0.49, − 0.73, and − 1.02 MPa), the content of MT was higher under NaCl stress than that of PEG-6000 and was relatively stable, or it could indicate that MT was an important endogenous substance for the salt tolerance of E. nutans seedlings under NaCl stress at a lower ambient water potential (Table 2).

In summary, the endogenous hormones of E. nutans seedlings to PEG-6000 responded differently to NaCl stress, and the endogenous hormones also interacted with each other based on the SEM and Mantel correlation analyses. Therefore, to explore the response of endogenous hormones of E. nutans to environmental water potential, further studies are needed to investigate the production and response of endogenous hormones.

Conclusions

In this study, we exposed E. nutans seedlings to controlled stressors of PEG-6000 and NaCl to examine the impacts of drought and salt stress under similar environmental water potentials. Our findings demonstrated differential responses in E. nutans seedlings, displaying higher tolerance to salt stress compared to drought stress. This was evident from the changes in plant biomass accumulation and physiological indices.

Our analysis of endogenous hormone levels indicated that fluctuations in the concentrations of GA3, GA7, 6-BA, 2.IP, TZ, KT, DHZ, IAA, IPA, IBA, NAA, and ABA were more pronounced under drought stress, emphasizing their roles in drought tolerance. In contrast, the levels of DHZ, TZR, SA, JA, and MT showed greater variations under salt stress, highlighting their significance in salt tolerance. These findings suggest that hormone regulation plays a crucial role in E. nutans adaptation to environmental stresses.

Future research should focus on further elucidating the molecular mechanisms governing these hormonal responses and their interactions, to better understand how E. nutans and similar species can be optimized for resilience against changing environmental conditions.