Melatonin Mediates the Regulation of Morphological and Anatomical Traits in Carex leucochlora under Continuous Salt Stress

Abstract

Soil salinity is one of the most critical factors limiting plant growth and development. Carex leucochlora is an important turfgrass species with a wide distribution in northern China that is highly sensitive to salt stress, which impairs its development. Recently, melatonin has emerged as a nontoxic biomolecule that regulates growth and enhances salt tolerance in plants. In this study, the mechanism of melatonin’s regulation of plant growth and anatomical characteristics in C. leucochlora seedlings under continuous salt stress was explored. Our results indicated that salt stress strongly suppressed plant growth and leaf cell activity, inhibited root morphology and root activity, and negatively affected leaf and root anatomic structures in the seedlings. Conversely, melatonin (150 μmol L⁻¹) pretreatment improved the detrimental effect of salt stress by restoring the morphology of the leaf, alleviating damage to the cell membrane, improving root activity, and altering the root architecture and plant growth attributes. Moreover, after 12 days of salt stress, anatomical observations of the leaf showed that the thickness of the leaf blade, vascular bundle area of the leaf main vein, vesicular cell area, thickness of the upper epidermis, and thickness of the lower epidermis were increased by 30.55, 15.63, 12.60, 16.76 and 27.53%, respectively, with melatonin under salinity. Melatonin treatment also showed an increase of 5.91, 7.59, 15.57, and 20.51% in epidermal thickness, vascular cylinder diameter, xylem vessel diameter, and pith cell diameter, respectively, compared with salt stress after 12 days. These results suggest that melatonin alleviated salt stress through augmenting seedling growth, leaf cell activity, and root characteristics, maintained the stability of anatomic traits to maintain chloroplast cell homeostasis, and also protected the vascular tissues to promote the radial transport of water and ions in the C. leucochlora seedlings. These modifications induced by the exogenous application of melatonin may help C. leucochlora to acclimate successfully to saline soils.

1. Introduction

Soil salinity is one of the most significant abiotic stresses hampering plant growth and development, posing a serious threat to agricultural production and human life [1]. Approximately 7% of the world’s total land area (1 billion hectares of land globally) is salt-affected [2]. Due to improper irrigation methods, unreasonable fertilizer application, and industrial pollution, the area of salinized land continues to increase, and it is predicted that by 2050, more than half of all arable land will be salinized [3]. High salinity is commonly caused by high concentrations of sodium (Na⁺) and chloride (Cl⁻) ions in the soluble fraction of the soil, resulting in both hyperionic and hyperosmotic conditions, which in turn impair the ability of plants to take up water and micronutrients [4]. This increase in oxidative stress ultimately causes decreases in photosynthetic rate and nitrogen metabolism, as well as increased osmolyte accumulation, resulting in an overall decrease in plant growth and cell apoptosis [5]. To tolerate the detrimental effects of salt stress, plants have developed various intrinsic adaptation strategies that include morphological, anatomical, physiological, and biochemical changes to assist their survival [6].

Plants can accumulate excessive reactive oxygen species (ROS) under the combined action of osmotic and ionic stress. Excessive ROS can cause cell damage to plants. Photosynthesis is an important physical and chemical process for plants that can be negatively influenced by ROS [7]. Salt stress damages chloroplast structure [8], and chloroplasts in mesophyll cells are the organelles most sensitive to salt stress [9]. For instance, salt stress causes the enlargement and disordered arrangement of the thylakoids, blurred boundaries between the grana and stroma lamella, and membrane damage or disappearance, or even disintegration [10,11]. At the morphological level, the thickness of the leaf lamina, mesophyll tissue, and phloem tissue and the diameter of the metaxylem vessels decrease under salt stress [12].

The rhizosphere environment is directly affected by salinity-induced ion imbalance [13]. This is mainly through the Na⁺ and K⁺ contents, which have been shown to negatively interfere with homeostasis via the replacement of K⁺ by Na⁺ in the root cytosol [14]. In response to high Na⁺ conditions, H⁺ pumps and Na⁺/K⁺ transporters contribute to cellular ion exchange and homeostasis as physiological adaptations [15]. Stress causes the root architecture and other aspects of the root phenotype to change [16]. Under saline conditions, the diameters of the epidermal cells and xylem components of the root are reduced [17], and the root vessel elements and epidermis and endoderm cells become thickened [18] to prevent excess Na⁺ accumulation. In addition, lipid peroxidation occurs in the root cells, which reduces root cell activity [19] and increases ROS [20]. In the roots, salt stress reduces the elongation rate [21], reduces the length of the roots and lateral root number [16], and interferes with gravity responses, inducing halotropism [22], thereby causing disordered root morphology [23].

Phytohormones have been innovatively used to increase plant adaptability and protect plants from adverse environmental conditions [24]. Melatonin (N-acetyl-5-methoxytryptamine), also known as pinealein, is an evolutionarily conserved molecule that, while considered an animal hormone, is also found in a large number of plants [25]. In plants, exogenous melatonin regulates the processes of growth, photosynthesis, and morphogenesis, and plays an important role in the response to drought [24,26], low/high-temperature [27,28], heavy metals [29], and salinity [30,31,32,33].

Melatonin enhances plant resistance to salt stress in two ways: one is through the direct clearance of ROS, while the other involves enhanced photosynthetic efficiency, antioxidant enzyme activity, and metabolite content, as well as the regulation of transcription factors related to stress [18]. Studies have shown that melatonin protects photosynthetic functioning, reduces osmotic stress and ion toxicity, improves the mesophyll cell structure, increases the abundance of chloroplasts [7], and improves the anatomical structure of plants under salt stress [34]. It is also considered an effective bio-stimulator and growth-promoting chemical that can accelerate coleoptile emergence in young seedlings and root growth [35]. Melatonin can alleviate the inhibition of salt stress by increasing the dry weight and fresh weight of the shoots and roots [33], modulate root system architecture [36], and alter root hormone levels [37]. At the same time, melatonin increases the H⁺-pump activities in the roots, thus promoting Na⁺ efflux and K⁺ influx, thereby maintaining K⁺/Na⁺ homeostasis and mitigating Na⁺ toxicity [15].

Carex leucochlora Bunge (Cyperaceae) is a perennial herbaceous plant that is one of the most ecologically important genera of perennial herbs distributed in temperate and cold regions around the world. It is used for lawn management, ecological protection, and feed production [38,39,40] and is characterized by early green recovery, a good color, strong treading resistance, an attractive growth habit, and has strong temperature adaptability. It is one of the main Carex species utilized in Chinese landscaping. There have been several studies on the seed dormancy and germination [41,42], classification [43], ecological diversity [44], and stress adaptability [39,45,46] of Carex. However, the effects of exogenous melatonin on alleviating salt stress damage in this plant are still elusive. No study has been conducted at the morphological and anatomical levels to understand the mechanisms of adaptation of C. leucochlora leaves and roots to salt stress, and furthermore, an investigation of the manner in which melatonin regulates the response to continuous salt stress in C. leucochlora is lacking. Therefore, in the present study, we used morphological, physiological, and anatomical approaches to gain insight into the melatonin-mediated regulation of C. leucochlora under salt stress. The specific objectives included the following: (i) examining the morphological integrity of seedlings supplemented with melatonin under continuous salt stress; (ii) elucidating the anatomical features of the leaves and roots supplemented with melatonin under continuous salt stress; (iii) evaluating the changes in root growth and architecture of C. leucochlora in response to persistent salt stress following melatonin pretreatment. These findings provide some insights for mitigating the negative effects of salt on the growth and development of C. leucochlora and may provide a promising strategy for promoting C. leucochlora establishment on salinized land.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The C. leucochlora variety ‘Four seasons’ (China accession No. S-SV-CL-006-2010) was used in this study and is commonly cultivated in the northern landscape of China, which was bred and cultivated at the Beijing Research and Development Center for Grass and Environment.

The 0.05 g seeds were sown in 10 cm × 6.5 cm × 8 cm (caliber × bottom diameter × height) plastic containers filled with culture medium containing peat soil and vermiculite (1:1 volume ratio). The experiment was performed in a growth chamber with 14 h of light at 25 ± 2 °C and 10 h of dark at 20 ± 2 °C, 60 to 70% relative humidity, and 500 μmol m⁻² s⁻¹ photosynthetically active radiation.

2.2. Melatonin and Salt Stress Treatments

The experiment was a randomized complete block design with four replications per treatment and fifteen pots of seedlings for each treatment were grown. The experiment had four treatments: plants (1) without NaCl and MT treatment (CK, control); (2) plants with MT treatment (MT, treated with 150 µmol L⁻¹ MT); (3) NaCl stress without MT treatment (S, treated with 150 mmol L⁻¹ NaCl); (4) NaCl stress + MT treatment (S + MT, pre-treated with 150 µmol L⁻¹ MT then treated with 200 mL 150 mmol L⁻¹ NaCl).

Carex leucochlora seedlings at 50 d after the seedling stage were sprayed with melatonin solution, and Tween 80 (1.0 mL L⁻¹) was added to the configured melatonin solution. Each spray was administered at 19:00 for 5 d consecutively. Each pot was sprayed with 5.0 mL of liquid. In order to choose the appropriate concentration for the MT treatment, five different concentrations (0, 100, 150, 200, and 250 µmol L⁻¹) were applied to the preliminary experiment. The control plants were watered with distilled H₂O (5.0 mL) alone. After the final spraying, the seedlings were irrigated with the NaCl solution (50 mL) every 3 d for 12 d. The first day before stress was recorded as 0 d. At 0, 3, 6, 9, and 12 d, the morphological traits and ultrastructure of the root and leaf were determined.

2.3. Withered Leaf Rate of Plant

The observational counting method was used to count the leaves of each pot of C. leucochlora treated, and the number of leaves with withered symptoms in 50% of the leaf area of each pot was calculated as a percentage of the total leaf number, which was recorded as the leaf wilt rate.

2.4. Relative Electrolyte Leakage

The relative electrolyte leakage (REL) was determined according to the method described by Zhang [47]. Approximately 0.2 g fresh leaf samples from each treatment were washed three times with distilled water and cut into 15 mm pieces, and then incubated in 20 mL ultrapure water for 6 h at 25 °C. After incubation, the electrolyte leakage was determined (EL₀) using a DDS-307A conductivity meter (INESA Scientific Instrument Co., Ltd., Shanghai, China). Subsequently, the solution was boiled (100 °C) for 30 min, and the electrolyte leakage of the cooled bathing solution was measured (EL₁). The REL was calculated by the formula: REL = EL₀/EL₁ × 100%.

2.5. Leaf Cell Relative Activity

Cell viability was detected by Evans blue staining [48,49]. The leaves of each treatment were detached and infiltrated in 0.25% (w/v) Evans blue solution in the dark for 24 h at 25 °C, and then the stained leaves were washed with distilled water and transferred to discoloring fluid (waterless ethanol: lactic acid: glycerol = 3:1:1), boiled in a water bath to remove the chlorophyll, and then photographed with a Nikon d7000 camera (Nikon Corporation, Tikyo, Janpan). The leaf staining area (A) and total leaf area (At) were measured by ImageJ analysis software, and the cell relative activity (V) was calculated: V = 1 − A/At × 100%.

2.6. Root Growth Analysis

The dry matter of the roots was measured after oven drying to a constant weight at 85 °C. Structural characteristics of the root samples were obtained by using an Epson Scanner V800 (Seiko Epson Corp., Tokyo, Japan) with WinRHIZO software (v4.0b, Regent Instruments Inc., Quebec, QC, Canada). The root length, root surface area, root average diameter, and root volume were measured.

2.7. Index of Root Activity

The root activity was recorded using the triphenyl tetrazolium chloride (TTC) method of Zhang et al. [50]. Root tip samples (0.5 g) were immersed in 10 mL of 0.4% (w/v) TTC and 10 mL sodium phosphate buffer (pH 7.0) under dark conditions for 4 h at 37 °C until the apical section turned completely white. The root samples were extracted with 5 mL of ethyl acetate. The extracted solution was transferred and fixed to 10 mL by adding ethyl acetate. The extracted solution from the root was measured at 485 nm, and the root activity was expressed as the TTC reduction intensity. Root activity = amount of TTC reduction (µg)/fresh root weight (g) × time (h).

2.8. Leaf and Root Anatomical Measurements

The leaf and root tissue samples were washed with water, following which the surface water was drained. The samples were then cut into small 0.2 cm segments, and the segments were fixed with FAA fixative solution (ethanol: formaldehyde: glacial acetic acid = 18:1:1). The samples were then washed in 50% ethyl alcohol, dehydrated in a normal butyl alcohol series, embedded in paraffin wax at the melting point of 56 °C, cut into sections with a thickness of 20 μm, double-stained with safranin/solid green, and then observed with a biological microscope (LEICA DM 2500, Leica Microsystems Inc., Buffalo Grove, IL, USA). The anatomical characteristics of the leaf and root were measured, and 10 readings were recorded using a micrometer eyepiece with five fields of view selected per sample.

2.9. Statistical Analysis

To determine the significance of the different treatments, an ANOVA was performed using SPSS software (20.0, SPSS, Inc., Chicago, IL, USA). Treatment means were compared using the Duncan’s multiple range test at the p ≤ 0.05 level for five independent biological experiments. OriginPro2019 (OriginLab Institute Inc., Northampton, MA, USA) was used to create graphs.

3. Results

3.1. Plant Growth Characteristics

The C. leucochlora seedlings were subjected to salt stress and examined for phenotypic foliar wilting (Figure 1a). Under salt stress treatment for 3 d, the C. leucochlora seedlings treated with melatonin or not showed no significant differences. However, after 6 days of salt treatment, the growth of the salt-treated plants (S) without melatonin decreased, the leaf tips withered and turned yellow, and the lawn quality decreased significantly when compared with CK. The withered leaf rate in melatonin treatments (S + MT) decreased by 59.45, 67.98, and 68.37% after 6, 9, and 12 days treatment, respectively, compared with salt stress. Therefore, the exogenous application of melatonin significantly restored the morphology of the leaf under salt stress.

To test the extent to which melatonin alleviated the degree of cell death, leaves treated with melatonin were subjected to salt stress and the cell viability was measured. There was no significant difference in the relative cell viability of the C. leucochlora seedling leaves in the MT treatment when compared with CK for the entire duration of the experiment (Figure 1c,d). In contrast, the salt stress treatment by 6 d resulted in intensely stained areas when compared to the control plants, and the relative activity of the leaf cells decreased significantly by 39.60%. However, under salt treatment, the melatonin-treated plants presented fewer blue stains than the non-treated seedlings, and the relative activity of the leaf cells increased significantly by 40.71%. With the extension of salt stress duration, the relative activity of the cells decreased. The S + MT treatment increased the cell activity by 1.62-fold and 4.29-fold, respectively, compared with salt treatment alone at 9 and 12 d (Figure 1c). The Salt × Time (S × T), Melatonin × T (M × T), and S × T × MT interaction for withered leaf rate and relative cell viability were all significant (p < 0.001) (Figure 1c,d).

3.2. Relative Electrolyte Leakage

The REL of the C. leucochlora seedlings remained low and stable under salt-free conditions (Figure 2). Salt stress led to a rapid increase in REL, and the longer the salt stress was applied, the higher the REL was, which increased by 4.92-fold (3 d) to 14.10-fold (12 d) compared with CK. Melatonin application significantly (p < 0.05) decreased REL for the entire duration of salt stress. When compared to the salt treatment, the REL of the S + MT treatment was reduced by 22.97–37.30% from 3 to 12 d. Therefore, under salt stress, melatonin application reduced membrane damage by modulating leaf REL in the C. leucochlora seedlings. The interaction term S × T, M × T, and S × T × MT for REL (p < 0.001) was significant.

3.3. Leaf Anatomical Structure

As shown in Figure 3, changes in the anatomical structure were observed in both the salt-stressed C. leucochlora seedlings and salt-stressed C. leucochlora seedlings treated with melatonin. After treatment for 3 days, the sponge tissue cells exhibited no obvious change; the chloroplasts were evenly distributed in the sponge tissue; the upper and lower epidermis were complete and arranged neatly and orderly; the chloroplasts were scattered in the mesophyll tissue (Figure 3a,b). However, when compared with CK, the thickness of the leaf blade under salt stress decreased non-significantly after 3 days of treatment (Figure 3c). From the 6th to 12th days of salt stress treatment, the vesicular cell shrank and became deformed, whereas the size of the vesicular cell in the S + MT treatment increased and the cell shape recovered when compared with the salt stress treatment (Figure 3a). Concurrently, under salt stress, the number of leaf sponge cells decreased and the cells were stretched from a regular circle to an irregular ellipse, the number of chloroplasts decreased, and the cell boundaries were unclear. Under S + MT treatment, the number of leaf sponge cells increased, the shape remained regular, and the number of chloroplasts in the sponge cells increased when compared with the salt treatment (Figure 3b).

With increasing exposure time to salinity stress (6 d to 12 d), the thickness of the leaf blade, vascular bundle area of the leaf main vein, vesicular cell area, thickness of the upper epidermis, and thickness of the lower epidermis decreased when compared with CK (Figure 3c–g).

The application of melatonin ameliorated the changes in the anatomical traits of the leaves. After 6–12 days of salt stress, melatonin application resulted in increases of 5.99–30.55, 0.46–23.79, 4.15–32.50, 7.56–20.87, and 2.70–20.53% in leaf blade thickness, vascular bundle area of the leaf main vein, vesicular cell area, upper epidermis thickness, and lower epidermis thickness, respectively (Figure 3c–g). The S × T, M × T, and S × T × MT interaction for leaf anatomical structure traits were all significant (p < 0.001) (Figure 3c–g).The changes in leaf anatomical structure under S + MT treatment could be attributed to the ability of the melatonin pretreatment to alleviate the deteriorative effect of salinity stress.

3.4. Root Characteristics

The root morphology of the C. leucochlora seedlings was significantly impacted by salt stress (

Table 1. Effects of the foliar application of melatonin on the root characteristics of C. leucochlora seedlings under salt stress. For each treatment day, different letters within a column indicate significant differences according to the Duncan’s multiple range test at p < 0.05 (n = 5). Mean ± standard error of five replicates. Significance levels are presented for each main effect or interaction. NS: not significant; *** p < 0.001; ** p < 0.01.

Day	Treatment	Root Dry Weight (mg·plant−1)	Root Length (cm)	Root Surface Area (cm2)	Root Average Diameter (mm)	Root Volume (cm3)
0 d	CK	5.05 ± 0.07 a	62.06 ± 0.18 a	4.20 ± 0.15 a	0.194 ± 0.001 a	0.017 ± 0.001 a
	MT	5.06 ± 0.11 a	63.62 ± 1.46 a	4.37 ± 0.09 a	0.197 ± 0.004 a	0.019 ± 0.002 a
	S	5.00 ± 0.18 a	61.65 ± 0.74 a	4.15 ± 0.11 a	0.195 ± 0.004 a	0.018 ± 0.002 a
	S + MT	5.10 ± 0.36 a	62.48 ± 1.62 a	4.37 ± 0.05 a	0.200 ± 0.003 a	0.018 ± 0.001 a
3 d	CK	5.67 ± 0.17 a	85.21 ± 0.47 ab	5.36 ± 0.04 a	0.206 ± 0.001 a	0.023 ± 0.001 a
	MT	5.72 ± 0.18 a	88.62 ± 0.52 a	5.48 ± 0.23 a	0.211 ± 0.006 a	0.024 ± 0.002 a
	S	5.46 ± 0.12 a	70.40 ± 1.55 c	4.79 ± 0.14 b	0.195 ± 0.003 b	0.022 ± 0.002 a
	S + MT	5.71 ± 0.17 a	82.89 ± 0.53 b	5.27 ± 0.03 a	0.203 ± 0.002 ab	0.024 ± 0.003 a
6 d	CK	8.38 ± 0.29 a	125.53 ± 2.06 a	6.11 ± 0.14 a	0.211 ± 0.001 a	0.030 ± 0.001 a
	MT	8.71 ± 0.17 a	127.31 ± 1.39 a	6.23 ± 0.05 a	0.226 ± 0.006 a	0.030 ± 0.001 a
	S	6.17 ± 0.21 c	87.10 ± 2.23 c	4.99 ± 0.11 c	0.198 ± 0.003 c	0.026 ± 0.002 a
	S + MT	7.44 ± 0.18 b	103.21 ± 3.85 b	5.67 ± 0.28 b	0.206 ± 0.005 b	0.028 ± 0.002 a
9 d	CK	10.39 ± 0.08 a	150.52 ± 3.51 a	8.62 ± 0.12 a	0.221 ± 0.002 a	0.045 ± 0.001 a
	MT	10.22 ± 0.17 a	154.67 ± 2.86 a	9.03 ± 0.14 a	0.224 ± 0.002 a	0.045 ± 0.001 a
	S	7.43 ± 0.14 c	98.30 ± 4.96 c	5.59 ± 0.27 c	0.203 ± 0.004 c	0.035 ± 0.002 b
	S + MT	9.67 ± 0.08 b	129.17 ± 6.43 b	7.27 ± 0.18 b	0.212 ± 0.002 b	0.043 ± 0.002 a
12 d	CK	12.22 ± 0.17 a	161.59 ± 4.42 a	10.08 ± 0.41 a	0.227 ± 0.003 a	0.055 ± 0.003 a
	MT	12.17 ± 0.08 a	163.39 ± 8.81 a	10.39 ± 0.24 a	0.231 ± 0.006 a	0.054 ± 0.002 a
	S	8.33 ± 0.08 c	99.80 ± 2.38 c	5.55 ± 0.19 c	0.186 ± 0.005 c	0.036 ± 0.001 c
	S + MT	10.61 ± 0.25 b	136.74 ± 0.59 b	7.83 ± 0.02 b	0.203 ± 0.003 b	0.043 ± 0.001 b
Salt (S)		***	***	***	***	***
Time (T)		***	***	***	***	***
M (Melatonin)		***	***	***	***	***
S × T		***	***	***	***	***
M × T		***	***	***	***	***
S × T × M		***	***	**	NS	NS

). Upon exposure to salt stress, the root dry weight had not changed significantly on the third day after stress, but decreased significantly (p < 0.05) on the 6th, 9th, and 12th day after salt stress. Meanwhile, when compared with the roots in CK, the root dry weight under salt stress decreased significantly by 26.42% (6 d), 28.50% (9 d), and 31.82% (12 d), respectively. Pretreatment with melatonin increased the root dry weight under salt stress significantly (p < 0.05) on the 6th to 12th day after stress, with an increase of 20.72 and 30.13%, respectively when compared to salt stress alone. The S × T, M × T, and S × T × MT interaction for root dry weight was significant (p < 0.001). This confirmed that salt stress inhibits the growth of the root system, and melatonin can effectively alleviate the inhibition of salt stress on the growth of the root system of C. leucochlora.

The root length, root surface area, and root average diameter under salt treatment were significantly (p < 0.05) lower than in the control on the third day after salt sress, and the root volume decreased significantly (p < 0.05) on the ninth day. With the prolongation of salt stress from the 3rd day to 12th day, the root length, root surface area, root average diameter, and root volume decreased significantly when compared with CK. In addition, the exogenous application of melatonin resulted in increases in root length of 17.73–7.02%, root surface area of 9.99–40.92%, root average diameter of 4.36–8.96%, and root volume of 7.58–21.91% when compared with salt treatment alone on the 3rd to 12th days. The interaction term S × T and M × T for root length was significant (p < 0.001). The S × T × MT interaction for root length (p < 0.001) and root surface area (p < 0.01) was significant, and was not significant for root average diameter (p = 0.732) and root volume (p = 0.0707). In general, the S + MT treatment altered the root architecture of the C. leucochlora seedlings under continuous salt stress.

3.5. Root Activity

During the entire experimental period, there was no significant difference in root activity between the CK and MT treatments (Figure 4). When compared with CK conditions, the root activity decreased significantly (p < 0.05) on the third day after salt stress and then decreased sharply by 18.04, 27.43, and 34.44% after 6, 9, and 12 days of salt treatment, respectively. Although the S + MT treatment did not significantly improve the root activity on the third day of salt stress, it did significantly stimulate root activity (p < 0.05) after 6 days, resulting in an increase in root activity of 15.52, 22.31, and 26.66% after 6, 9, and 12 days treatment, respectively, compared with salt stress alone. The S × T, M × T, and S × T × MT interactions for root activity were all significant (p < 0.001). Taken together, these results suggest that the regulation of melatonin pretreatment on the root activity of C. leucochlora under salt stress was strongly induced after 6 days.

3.6. Root Anatomical Structure

To assess the root anatomical changes in C. leucochlora during salt stress under exogenous melatonin, we analyzed cross-sections of the roots of plants exposed to different treatments (Figure 5).

On the third day of salt stress, the cortex layer cells of the salt-stressed seedlings were deformed, and the size and shape were uneven. With the extension of salt stress duration (6 to 12 days), the cortex layer cells of the S treatment group were severely deformed, some cells died, the degree of damage to the cortex layers increased, the number of dead cells in the cortex increased, and the number and size of cavities both increased (Figure 5a). With increasing exposure to salinity stress (3 to 12 days), the epidermal thickness, xylem vessel diameter, and pith cell diameter increased first then decreased, and the vascular cylinder diameter decreased. These four traits were 31.64, 33.55, 19.54, and 31.99% lower than in CK, respectively, after 12 days of salt stress (Figure 5b,e).

The application of melatonin could significantly reduce the effect of salt stress on the root anatomical structure. After 12 days of salt stress, melatonin application resulted in increases of 5.91, 7.59, 15.57, and 20.51% in epidermal thickness, vascular cylinder diameter, xylem vessel diameter, and pith cell diameter, respectively, when compared with salt stress. The S × T and M × T interaction for root anatomical structure traits was significant (p < 0.001). The S × T × MT interaction for epidermal thickness (p < 0.001), vascular cylinder diameter (p < 0.01), xylem vessel diameter (p < 0.05), and pith cell diameter (p < 0.001) was significant. The changes in root anatomical structure under the S + MT treatment could be attributed to the ability of melatonin pretreatment to alleviate the deteriorative effect of salinity stress.

4. Discussion

Excessive salinity occurs under natural and agricultural conditions and can impose both osmotic and ionic stresses, which limit the ability of cells to take up water from the environment and cause cytoplasmic and organellar toxicity [51,52]. Salt stress can significantly impact plant growth and physiological and biochemical traits [53]. In recent years, melatonin has been proposed as a master plant growth regulator that helps to boost plant growth and productivity under abiotic stress conditions [24,33]. We discovered for the first time herein that melatonin regulates C. leucochlora seedling growth under saline conditions, with a melatonin concentration of 150 μmol L⁻¹ being most effective for protecting against salinity-induced stress. When compared with the salt stress treatment alone, pretreatment with 200 and 250 μmol L⁻¹ exogenous melatonin and then salt stress treatment can not reduce the degree of wilting of C. leucochlora seedling leaves (data not shown). Therefore, the effect of melatonin was found to be dose-dependent [49]. Our results demonstrated that melatonin mitigates the negative effects of salt stress on the growth characteristics and maintains the morphological integrity of C. leucochlora seedlings. This is consistent with earlier studies, whereby melatonin-treated field crops [32,33], horticultural plants [36,45], and turfgrasses [27,54] were found to possess enhanced salt tolerance.

Changes in EL can reflect the extent of damage to plant membrane permeability in response to environmental stimuli [55]. Melatonin pretreatment significantly inhibited the increase in EL under salt stress in C. leucochlora herein (Figure 2), indicating that melatonin could effectively alleviate the damage of salt stress to the cell membrane, as also reported for cucumber [47] and bermuda grass [Cynodon dactylon (L.). Pers.] [54].

Photosynthesis is indispensable for plant growth and development [7]. Salt stress can destroy the chloroplast structure and decrease the contents of chlorophyll and osmotic adjustment substances [7,31], which has an adverse effect on the bioenergetic process of photosynthesis [18]. Salinity stress had a considerable negative impact on leaf anatomical structure, as indicated by the decreased leaf blade thickness, reduced number of chloroplasts in the mesophyll cells, severe deformation of the upper and lower epidermal cells, and disordered arrangement of sponge tissue cells (Figure 3). These results are supported by previous reports on wheat [12], amaranth (Amaranthus caudatus L.) [56], and kallar grass (Leptochloa fusca (L.) Kunth) [57]. The C. leucochlora plants treated with melatonin possessed thicker leaves and an improved vesicular cell area during the entire stress period, suggesting that the balancing of K⁺/Na⁺ levels and the adjustment of osmolyte accumulation by melatonin pretreatment ameliorated the impacts of salinity stress [15]. Melatonin also promoted the vascular bundle area of the C. leucochlora seedlings under salt treatment (Figure 3d). These anatomical changes may benefit water and solute transport capacity. These results indicate that melatonin treatment maintained the leaf structure to reduce the inhibition of photosynthesis and has protective effects on chlorophyll damage under salt stress.

The root is the first organ to perceive salt stress, and its structural characteristics largely determine plant’s adaptability to the soil environment [58]. Under high salt conditions, root elongation, lateral root initiation, and the number of lateral roots were inhibited and plant growth was decreased in Arabidopsis thaliana L. [23], which was mediated by auxin redistribution in the roots, which modulates root architecture plasticity [59,60]. Our study results illustrated that salt treatment negatively affected root morphological traits by decreasing the root dry weight, root length, root surface area, and root average diameter. Conversely, the pretreatment of C. leucochlora roots with melatonin evidently enhanced the root characteristics (Table 1), contributing to improved plant growth. Our findings are consistent with those obtained for tomato [36,61] and canola (Brassica napus L.) seedlings [35] under salt stress and other abiotic stresses such as drought⁶ and heavy metals [62]. The current study also demonstrated that melatonin treatments increased the root viability under salinity stress (Figure 4). Thus, as reported previously, exogenously applied melatonin can ameliorate salinity stress in roots.

Root anatomy is a crucial determinant of both tissue composition and the proportion of root tissue that is metabolically active [63]. In the root, high salt stress reduces the epidermis and endoderm cells and root vessel elements as a way to ameliorate the deleterious effects of excess Na⁺ in this organ [64]. The epidermal thickness, xylem vessel diameter, and pith cell diameter of the C. leucochlora seedlings increased first then decreased, and the vascular cylinder diameter decreased with an increased salt stress duration, which may constitute a defensive attribute under salinity conditions [17]. In contrast, melatonin treatment helped to maintain root anatomical stability under salt stress (Figure 5). In this context, vascular cylinder and xylem vessels contribute to the conduction of water to the upper organs, and melatonin contributes to the prevention of the loss of conductive cell functionality. Moreover, epidermal thickness represents a mechanical barrier in the radial transport of water and ions to protect vascular tissues [65], and increased Na⁺ concentration under salt stress causes plasmolysis and reductions in the protective tissues [14]. In this context, melatonin treatment increased the epidermal thickness of the C. leucochlora seedlings under salt stress. In A. thaliana, melatonin promotes primary root growth in an indole-3-acetic acid (IAA)-dependent manner, with changes in gene expression following melatonin and IAA application being co-regulated [66], which modulates the root ultrastructure to adaptation salt stress.

5. Conclusions

The present study explored the collective decline in salt stress resilience in C. leucochlora as indicated by foliar wilting, reduced morphological integrity, decreased cell activity in the leaves and roots, damaged root morphology, and an impaired ultrastructure under continuous salt stress. Per contra, the stress induced by salt was effectively mitigated by the exogenous application of melatonin, which resulted in delayed foliar wilting, enhanced leaf and root cell activity, improved root architecture, and the recovery of ultrastructure (Figure 6). The results of this study provide, for the first time, conclusive evidence on the protective role of exogenous melatonin in improving salt tolerance in C. leucochlora seedlings. Our research provides evidence for melatonin-mediated salt tolerance and has crucial applications for improving the growth of horticultural crops on salinity soils.