Morphological and physiological responses of two willow species from different habitats to salt stress

Plant salt tolerance is a complex mechanism, and different plant species have different strategies for surviving salt stress. In the present study, we analyzed and compared the morphological and physiological responses of two willow species (Salix linearistipularis and Salix matsudana) from different habitats to salt stress. S. linearistipularis exhibited higher seed germination rates and seedling root Na+ efflux than S. matsudana under salt stress. After salt treatment, S. linearistipularis leaves exhibited less Na+ accumulation, loss of water and chlorophyll, reduction in photosynthetic capacity, and damage to leaf cell structure than leaves of S. matsudana. Scanning electron microscopy combined with gas chromatography mass spectrometry showed that S. linearistipularis leaves had higher cuticular wax loads than S. matsudana leaves. Overall, our results showed that S. linearistipularis had higher salt tolerance than S. matsudana, which was associated with different morphological and physiological responses to salt stress. Furthermore, our study suggested that S. linearistipularis could be a promising tree species for saline-alkali land greening and improvement.

germination rate, germination energy, and germination index of S. linearistipularis seeds was significantly higher than that of S. matsudana under treatments of 150 and 200 mM NaCl ( Fig. 1a-d). Under 200 mM NaCl treatment, S. linearistipularis seeds still had a 25% germination rate, whereas S. matsudana seeds were completely unable to germinate. However, under untreated control conditions, their germination rate was similar (about 78%; Fig. 1a,b).
Furthermore, Na + and K + flux in roots of S. linearistipularis and S. matsudana seedlings was compared by noninvasive micro-test technique (NMT) (Fig. 2a), and the results showed that NaCl treatment caused the increase of Na + and K + efflux rate in both seedling roots and that the Na + and K + efflux rate of S. linearistipularis roots was significantly higher than that of S. matsudana under salt stress. However, there was no significant difference between the Na + and K + efflux rates of the two species under control conditions in which both species exhibited weak Na + and K + efflux (Fig. 2b,c). These results suggested that the S. linearistipularis has higher seed germination rate and seedling root Na + and K + efflux capacity under salt stress than those of S. matsudana. Na + and K + content and water loss of two willow species leaves under salt stress. The effects of salt stress on the seedlings growth of two willow species were compared. After NaCl treatments (150 and 200 mM), the Na + content in S. linearistipularis leaves was lower than that in S. matsudana leaves, while the K + content was slightly higher than that in S. matsudana leaves (Fig. 3a,b). Furthermore, S. linearistipularis leaves exhibited less reduction in fresh weight and maximal photochemical efficiency (Fv/Fm) than S. matsudana leaves, but they do not showed difference in dry weight (Fig. 3c-f). These results suggested that the S. linearistipularis leaves has less Na + accumulation and water loss under salt stress than those of S. matsudana.
Photosynthetic parameters and leaf ultrastructure of two willow species leaves under salt stress. In order to investigate the difference in salt tolerance between the two willow species, their leaf mor- Comparison of seed germination rates between S. linearistipularis (Sl) and S. matsudana (Sm) under normal and salt stress conditions. (a) Seeds of Sl and Sm were germinated on filter paper containing aseptic water (control) or NaCl solution (100, 150, and 200 mM) for 4 days, and their germination rate (8 days) (b), germination energy (3 days) (c), and germination index (8 days) (d) were calculated. The asterisk indicates significant difference (**p < 0.01; Student's t test). The error bar indicates SE (n = 6).

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| (2020) 10:18228 | https://doi.org/10.1038/s41598-020-75349-2 www.nature.com/scientificreports/ phology under salt stress was compared. After NaCl treatments (200, 300, and 400 mM), the area of S. matsudana leaves losing green color was generally larger than that of S. linearistipularis leaves (Fig. 4a). The analysis of relative chlorophyll (Chl) content showed that the decrease of Chl in S. matsudana leaves was significantly higher than that in S. linearistipularis leaves after NaCl treatment (Fig. 4b). Photosynthesis parameter measurements showed that the Fv/Fm value of S. matsudana leaves was significantly lower than that of S. linearistipularis leaves after NaCl treatments with more than 200 mM (Fig. 4a,c). These results suggested that the damage of salt to leaf photosynthetic capacity was significantly higher in S. matsudana than that in S. linearistipularis. Photosynthetic capacity is directly related to chloroplast structure and function. Under salt stress, the ultrastructure of cells in S. linearistipularis and S. matsudana leaves, especially particularly chloroplasts, was compared by TEM. TEM images showed similar healthy cellular structures in S. linearistipularis and S. matsudana leaves under control conditions ( Fig. 5a-d). After NaCl (300 mM) treatment, S. linearistipularis leaf cell structure remained unchanged, whereas S. matsudana leaf cell structure was almost completely damaged ( Fig. 5e-h). In S. matsudana leaf cells, we observed severe cytoplasmic wall separation, damaged chloroplast envelopes (CE) and thylakoid membranes (TM), and diffuse plastoglobuli (Fig. 5g,h). These results suggested that the damage of salt to leaf cell ultrastructure in S. matsudana was more serious than that of in S. linearistipularis.
Leaf crystal patterns and thickness of cuticular wax in two willow species leaves. As salt damage to leaf cell structure may be related to the permeability of leaf surface, we observed and compared the adaxial (upper) and abaxial (lower) surfaces of the two willow species leaves. SEM images showed that the adaxial surface of both S. linearistipularis and S. matsudana leaves has a waxy structure, and the waxy structures were highly similar in shape and density (Fig. 6a,b). However, the abaxial surface of both leaves was smooth (Fig. 6c,d).
Water adhesion analysis showed that on the adaxial surface of S. linearistipularis leaves, less water droplets were formed by the same volume of water than S. matsudana leaves (Fig. 7a), and therefore we speculated that the thickness of the wax layer on leaf surface differed between the two species. Furthermore, cross sections of leaves were observed using cryo-SEM, and the results showed that the willows had typical bifacial leaves ( Fig. 7b) with epidermal waxes accumulated on the adaxial surface in the leaves of both species (Fig. 7c,d). A large number of our observations found that the thickness of the wax layer on the adaxial surface of S. linearistipularis leaves was generally higher than that of S. matsudana leaves (Fig. 7e,f). These results suggested that the crystal pattern of the cuticular wax of the two willow leaves was highly similar, but the thickness of the wax differed between the two investigated species.
Components and loads of cuticular wax in two willow species leaves. Cuticular wax components from the leaves of the two willow species were extracted and analyzed. GC-MS revealed that the cuticular wax components of the two leaves were constituted of many chemical compounds, including fatty acids, alcohols, and alkanes (Fig. 8). These major components were identified by corresponding retention times and similarity matching scores (more than 600; Table 1). The results of the principal component analysis showed that the cuticular wax loads of the two leaves were well distinguished in content (Fig. 9a). Statistical analysis showed that the cuticular wax load of S. linearistipularis leaves was higher than that of S. matsudana leaves (Fig. 9b).

Discussion
Wild species inhabiting natural saline habitats possess genetic variations which are the basis of the evolution of salt tolerant populations [15][16][17] . S. linearistipularis is a woody plant naturally distributed in the saline-alkaline lands with high salinity in the Songnen plain of northeast China, showing its strong salt adaptability 8 . In the present study, we investigated the morphological and physiological characteristics associated with salt tolerance in S. linearistipularis and compared them to those of S. matsudana. Under salt stress, S. linearistipularis exhibited Figure 2. Comparison of root Na + and K + efflux rate between S. linearistipularis (Sl) and S. matsudana (Sm) seedling under normal and salt stress conditions. (a) Morphology and site of root monitored by NMT. Mean Na + (b) and K + (c) efflux rate from the roots of Sl and Sm seedlings (7-day-olds) after 12 h of incubation in aseptic water (control) or NaCl solutions (50 and 100 mM). The asterisk represents a significant difference (*p < 0.05; Student's t test). The error bar indicates SE (n = 6).

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| (2020) 10:18228 | https://doi.org/10.1038/s41598-020-75349-2 www.nature.com/scientificreports/ higher seed germination rate and higher seedling root Na + efflux capacity than that of S. matsudana (Figs. 1, 2). Similarly, the Na + efflux in roots of salt tolerant P. euphratica was significantly higher than that of salt sensitive P. popularis under salt stress 12 . Furthermore, S. linearistipularis leaves had less Na + accumulation under salt stress than S. matsudana leaves (Fig. 3a). Higher Na + efflux in roots may reduce Na + accumulation and its toxic effects under salt stress. Limiting the entry of salt (mainly Na + ) by the roots and maintaining lower Na + accumulation in tissues or cells is one of the main salt tolerance strategies evolved by plants 18,19 . These results suggested that the more active root Na + efflux capacity and less leaf Na + accumulation of S. linearistipularis seedlings under salt stress contributed to its salt tolerance.  www.nature.com/scientificreports/ Salt also causes injuries of the young photosynthetic leaves and accelerates their senescence 19 . In the present study, salt treatment caused a decrease in Chl content and photosynthetic capacity (indicated by Fv/Fm parameters) in the leaves of both willow species; however, S. linearistipularis leaves maintained higher Chl content and Fv/Fm value compared to those of S. matsudana leaves after salt treatment (Fig. 4). Furthermore, TEM analysis showed that the damage to S. linearistipularis leaf cell structures caused by the salt treatment, particularly, the damage to the photosynthetic membrane structures in chloroplasts, was significantly lower than that to S. matsudana leaves (Fig. 5e-h). These results suggested that less damage to leaf cell structure, chlorophyll loss, and reduction in photosynthetic capacity of S. linearistipularis leaves under salt stress contributed to its salt tolerance. However, the response of isolated leaves to salt may be different from that of living plants. Therefore, the morphological and physiological responses of leaves from two living willow plants under salt stress need further investigation and comparison.
Salt stress causes osmotic stress, resulting in the loss of water from plant leaves 20 . Plant cuticular waxes play a crucial role in limiting non-stomatal water loss in leaves 21 . In the present study, SEM analysis showed that the cuticular waxes were present on the adaxial surfaces of the leaves of both willow species, and the waxes had highly similar crystal patterns (Fig. 6a,b). However, cryo-SEM analysis showed that the thickness of the cuticular waxes in S. linearistipularis leaves was generally higher than that in S. matsudana leaves (Fig. 7e,f), which was also confirmed by GC-MS analysis (Fig. 9). Cuticular wax load was found to be negatively correlated with leaf water loss rate 22,23 . Fresh weight measurements showed that the water loss ratio of S. linearistipularis leaves under salt stress was lower than that of S. matsudana leaves (Fig. 3c). These results suggested that higher cuticular wax loads of S. linearistipularis leaves than those of S. matsudana leaves contributed to its higher salt tolerance. Different environmental conditions can affect the distribution and chemical composition of cuticular waxes in plants [24][25][26] . Thus, we speculated that the saline-alkali conditions affected the cuticular wax loads in S. linearistipularis leaves. Studies have shown that several species of the genus Salix differ in cuticular wax loads, but have highly similar wax composition 27,28 .
Overall, our study showed that compared to S. matsudana, S. linearistipularis has higher salt tolerance, which is associated with higher root Na + efflux, less leaf Na + accumulation, better maintenance of leaf cell structure and photosynthetic capacity, and higher cuticular wax load under salt stress conditions. Our results suggest that S. linearistipularis could be a promising tree species for saline-alkali land greening, improvement, and phytoremediation practices.  www.nature.com/scientificreports/ Seed germination test. Fifty seeds from two willows were sown on plates with filter paper containing aseptic water (control) or NaCl solutions of different concentrations (100, 150 and 200 mM). These seeds were cultured for 8 days at 22 °C before measuring seed germination rate, germination energy, and germination index. The experiment was replicated four times.

Materials and methods
Measurement of Na + and K + efflux. Seedlings grown in aseptic water were used for the measurement of Na + and K + efflux. Hydroponic seedlings (7-day-olds) were exposed to aseptic water (control) or NaCl solution (50 and 100 mM) for 12 h, and root segments were immobilized in the measuring solution (0.1 mM KCl, 0.1 mM CaCl 2 , 0.1 mM MgCl 2 , 0.5 mM NaCl, and 0.3 mM MES, pH 5.8) in order to measure the Na + flux. Net fluxes of Na + and K + were measured using the non-invasive micro-test technique (NMT100 Series, YoungerUSA LLC, Amherst, MA, USA) as described in 12,29 .

Measurement of chlorophyll content and chlorophyll fluorescence parameters. The leaves
were shaped into leaf discs (1 cm 2 ) which were immediately immersed in aseptic water (control) or NaCl solutions of different concentrations (50, 100, 200, 300 and 400 mM) for 48 h. Chlorophyll (Chl) was extracted from the leaf samples with 80% ice-cold acetone. The absorbances of Chl a (646 nm) and Chl b (663 nm) were determined using a UV/Vis spectrophotometer. The total Chl content was calculated as the sum of Chl a and Chl b. The maximal photochemical efficiency (Fv/Fm), minimal fluorescence yield (F 0 ), and maximal fluorescence yield (Fm) were measured using an Imaging-PAM Chlorophyll Fluorometer (Walz, Germany) as described in 30 .  Scanning electron microscopy (SEM and cryo-SEM). SEM and cryo-SEM analyses were performed as previously described 22 . For SEM analysis, leaf samples were collected, fixed with glutaraldehyde buffer, and gradually dehydrated using alcohol. The leaf samples were then dried to the critical point using liquid CO 2 and sputter coated with an electrically conductive gold layer before being imaged by SEM (Hitachi SU-8010, Tokyo, Japan) at 5 kV. For cryo-SEM analysis, leaf samples were sprinkled onto a perforated aluminum stub and plunged into liquid nitrogen slush (− 210 °C). The frozen samples were transferred to a cryo system (PP3010T; Quorum Technologies, Lewes, UK), sputter coated with platinum, transferred to the SEM cold stage, and examined at − 140 °C at a beam voltage of 5 kV and probe current of 10 mA.
Wax extraction. Leaf samples were placed into a 50 mL tube and 30 mL pre-cold extraction mixture (chloroform) was added. The samples were vortexed for 30 s and ultrasonicated in water for 30 min at 60 ± 5 °C. The samples were then taken out of the tubes and 20 μL of internal standard (adonitol, 0.5 mg mL −1 stock) was added to each tube. The samples were nitrogen blow-dried and reconstituted in 5 mL of chloroform by sonication on ice for 5 min. After centrifugation (4 °C, 10 min, 5000 rpm), 500 μL of the supernatant was transferred to a new tube. In order to prepare the Quality control (QC) sample, 150 μL of each sample was taken and these samples were combined.
After evaporation in a vacuum concentrator, 50 μL of methoxyamination hydrochloride (20 mg mL −1 in pyridine) was added and incubated at 80 °C for 30 min, and derivatization was achieved by dissolving the samples in 70 μL of BSTFA reagent (1% TMCS, v/v) at 70 °C for 1.5 h. The samples were gradually cooled to room temperature, and 5 μL of FAMEs (in chloroform) was added to the QC sample. All samples were analyzed by gas chromatograph coupled with a time-of-flight mass spectrometer (GC-TOF-MS) 32 .

GC-TOF-MS analysis.
GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph coupled with a time-of-flight mass spectrometer as previously described 32 . The system utilized a DB-5MS capillary column. A volume of 1 μL of sample aliquot was injected in splitless mode. Helium was used as the carrier gas, the front inlet purge flow was 3 mL min −1 , and the gas flow rate through the column was 1 mL min −1 . The initial temperature was kept at 50 °C for 1 min, then raised to 310 °C at a rate of 10 °C min −1 and kept at 310 °C for 8 min. The injection, transfer line, and ion source temperatures were 280 and 250 °C, respectively. The energy was − 70 eV in electron impact mode. The mass spectrometry data were acquired in full-scan mode with the m/z range of 50-500 at a rate of 12.5 spectra per second after a solvent delay of 6.25 min.
Data preprocessing. Raw data analysis, including peak extraction, baseline adjustment, deconvolution, alignment, and integration, was performed with Chroma TOF software (V 4.3x, LECO) 33 , and LECO-Fiehn Rtx5 database was used for metabolite identification by matching the mass spectrum and retention index. Finally, the peaks detected in less than half of QC samples or RSD > 30% in the QC samples were removed 34 .