Different Responses to NaCl vs. NaHCO₃ Stress in Three Limonium Species: Linking Seed Phenotype to Physiological Tolerance

Abstract

Soil salinization severely restricts vegetation restoration in Northwest China. Native Limonium plants, capable of naturally colonizing saline-alkalisaline–alkali wasteland, are potential germplasm for low-cost ecological restoration. This study focused on three wild Limonium species (Limonium aureum, Limonium bicolor, Limonium gmelinii) in Gansu Province. In this study, we integrated seed phenotypic diversity with stress tolerance. We then investigated seed germination indices (e.g., germination rate, energy, vigor index) and seedling physiological–biochemical indices of three Limonium species under 0, 100, 200, 300 mM NaCl and NaHCO₃ stress. These indices included leaf and root Na⁺ and K⁺ contents, chlorophyll a and b and carotenoid contents, and malondialdehyde (MDA), proline, soluble sugar, and soluble protein contents, plus SOD and CAT activities. Results showed seed area and thickness were key to germination performance, with L. aureum having the largest and thickest seeds and strongest germination potential. The onset concentration of significant inhibition for salt/alkali was 200 mM. At the seedling stage, L. aureum performed best at 100–200 mM, while all three were damaged at 300 mM. Principal component analysis indicated that L. aureum had the highest comprehensive scores under both NaCl and NaHCO₃ stresses, while L. bicolor and L. gmelinii presented distinct stress-specific adaptation differences. Thus, L. bicolor is recommended for salt-dominated soils and L. gmelinii for alkaline environments, and L. aureum can be used for mildly heterogeneous habitats. This study clarifies inter-species differences under stress, providing a direct theoretical basis for ecological restoration in saline–alkali areas.

1. Introduction

Soil salinization has become a global ecological issue, with its severity and affected area increasing annually, significantly restricting global agricultural development [1,2,3]. It is estimated that by 2050, nearly 50% of the world’s arable land will be impacted by salinization, characterized by high electrical conductivity, reduced water potential, and excess ionic salts, which poses a substantial threat to agricultural productivity [4]. In recent years, the interaction between climate change and human activities has led to a decline in plant biodiversity and productivity, creating significant challenges for the management and restoration of saline–alkaline lands [5]. To address these challenges, various restoration methods have been proposed and implemented, including irrigation and drainage [6], the application of soil amendments [7], and the protection and restoration of halophyte communities [8]. Among these, the use of salt-tolerant plants for phytoremediation has proven to be a sustainable and efficient approach. Phytoremediation, as a green governance technology, utilizes natural processes through mechanisms such as absorption, degradation, and fixation by plants, offering a low-cost and safe solution for environmental remediation. It is an important method for dealing with soil pollution and restoring sustainable ecosystems. Studies demonstrate that leveraging the ecological benefits of halophytes-including their capacity to ameliorate saline soil physicochemical properties, reduce soil salinity, and enhance soil fertility and biodiversity-represents a key strategy for promoting sustainable land use and ecological restoration under salt stress conditions [9,10].

Recent advances in halophyte physiology have fundamentally reshaped the understanding of salt tolerance, which is now recognized not as passive endurance but as a dynamic and integrated adaptive strategy operating across cellular, metabolic, and systemic levels. Central to this resilience is the precise maintenance of cytosolic ion homeostasis. Halophytes actively restrict Na⁺ influx at the root-soil interface and rapidly sequester excess Na⁺ into vacuoles through tonoplast-localized NHX (Na⁺/H⁺ exchanger) antiporters, thereby preserving low cytosolic Na⁺ concentrations that are essential for enzymatic function and metabolic stability [11]. Concurrently, these plants accumulate compatible solutes such as proline, glycine betaine, and soluble sugars. These compounds serve dual roles: they restore cellular osmotic balance and stabilize protein structures while also acting as reactive oxygen species (ROS) scavengers [11]. This osmotic adjustment is tightly coordinated with a robust antioxidant defense system that includes enzymatic components such as superoxide dismutase, catalase, and ascorbate peroxidase, as well as non-enzymatic antioxidants like ascorbate and carotenoids. Together, these elements mitigate oxidative damage to membranes, chloroplasts, and photosynthetic pigments under saline–alkaline stress [12]. Importantly, these integrated physiological responses often confer cross-tolerance to other abiotic stresses, by enhancing stomatal regulation, water-use efficiency, and cellular structural integrity. This capability represents a key evolutionary advantage in fluctuating saline–alkaline environments [13]. Collectively, these mechanisms illustrate how halophytes not only survive but thrive in otherwise hostile habitats.

Despite these sophisticated adaptations, the early life stages of halophytes, particularly seed germination and seedling establishment, remain highly sensitive to saline–alkaline stress. Saline and alkaline stress, as a major abiotic stress, negatively affects seed germination and seedling establishment through osmotic stress and ion toxicity. Saline stress is primarily caused by neutral salts such as NaCl and Na₂SO₄, with a pH typically ranging from 7 to 8. Alkaline stress, on the other hand, is mainly induced by alkaline salts such as NaHCO₃ and Na₂CO₃, which generally have a pH greater than 8.5 and impose additional high-pH and bicarbonate/carbonate-mediated cellular damage [14,15,16,17]. However, numerous studies show that alkaline salt stress is often more harmful to plants than neutral salt stress [16,18]. Previous studies have confirmed that seed germination and seedling growth directly determine subsequent plant establishment and survival in saline–alkaline lands [19], making research on their stress resistance during these early stages crucial for optimizing vegetation restoration practices [20]. However, significant interspecific differences exist in germination characteristics and stress tolerance among halophytes, which may stem from variations in metabolic pathways, cellular physiological regulation efficiency (e.g., ion transporter activity, antioxidant enzyme capacity), or gene expression patterns [21]. Unlike previous studies that mostly focused on single halophyte species and single stress types, this research simultaneously examines the responses of three closely related species, L. aureum, L. bicolor, and L. gmelinii, to salt and alkali stresses during the seed germination and seedling growth stages. Additionally, it analyzes these in combination with seed phenotypes. Based on this approach, this study reveals the correlation between the seed morphology and stress resistance of the Limonium genus. Furthermore, it helps to gain an in-depth understanding of the strategies employed by halophytes to cope with saline–alkali environments during their early development stages, thus providing a new perspective for understanding their adaptation mechanisms.

Phenotypic traits are the most direct manifestation of plant growth diversity, influenced by both genetic characteristics and environmental factors [22]. The study of phenotypic variation not only helps reveal the mechanisms and patterns of plant responses to environmental factors but also plays a crucial role in the collection, conservation, and evaluation of plant genetic resources [23]. Plant phenotypic traits continuously generate new phenotypes in response to changes in environmental conditions, providing valuable insights into plant adaptability and the identification and selection of new varieties [24,25]. Particularly in extreme environments such as saline–alkaline soils, plant phenotypic traits, including root systems, leaves, and physiological characteristics, directly respond to saline–alkaline stress by altering morphological structures and physiological activities to enhance salt tolerance, thus ensuring survival and reproduction [25]. Importantly, seed morphological and structural traits (e.g., shape, size, coat thickness) can predict germination responses and early seedling growth, and may even affect community renewal success [26]. Specifically, seed phenotypic traits of wild native species (such as coat thickness, area, length) correlate significantly with germination and seedling establishment [27]. Therefore, in-depth study of seed phenotypic diversity is of great significance for understanding plant adaptation mechanisms in saline–alkaline environments, as well as providing a theoretical foundation and practical value for the conservation and utilization of halophytic plant genetic resources [28].

L. aureum, L. bicolor and L. gmelinii, typical halophytes of the family Plumbaginaceae, are widely distributed in typical saline–alkaline habitats such as deserts, Gobi, salt flats, and lake basins, and are important halophytic plant resources in the arid regions of northwest China. These species not only exhibit strong salt tolerance but also play a pioneering role in ecological restoration of saline–alkaline soils [29]. In addition, Limonium species are highly valued for their ornamental flower clusters, and are widely used in the cut flower industry and desert landscape beautification, serving both ecological and economic purposes [18]. Although existing studies have extensively explored the salt tolerance mechanisms of Limonium species under neutral salt conditions such as NaCl and Na₂SO₄, the response mechanisms under alkaline salt conditions remain relatively understudied [30]. Unlike NaCl, which mainly induces osmotic and Na⁺ toxicity, NaHCO₃ additionally causes high-pH and bicarbonate-mediated damage to membranes, nutrient balance, and photosynthesis [31]. Particularly in the typical saline–alkaline complex environments of the northwest, the mechanisms of stress tolerance in Limonium species are rarely studied systematically, which limits the application and promotion of relevant findings in the restoration of saline–alkaline vegetation and the utilization of halophytic plant resources [32]. Previous studies have shown that halophytes from different genera, such as Suaeda salsa, Aeluropus sinensis, and Phragmites communis, exhibit diverse tolerance strategies under salt and alkali stresses, and thus occupy different ecological niches [21]. Based on this premise, we speculate that L. aureum, L. bicolor, and L. gmelinii, as different species within the same genus, may adopt different adaptation strategies in saline–alkali environments and display species-specific physiological and ecological niche characteristics under different types of saline–alkali soils.

This study used L. aureum, L. bicolor and L. gmelinii as plant materials to investigate the effects of saline and alkaline stress at different concentrations on the growth, biochemical, and physiological adaptation mechanisms of these species. The research focused not only on seed germination and seedling growth but also on the changes in morphological, physiological, and biochemical indicators. Specifically, we measured the germination rate (GR), germination potential (GP), germination index (GI), and Vigor Index (VI) of seeds, as well as the fresh weight (FW), dry weight (DW), and total biomass (TB) of roots and leaves, chlorophyll content, ion content, osmotic regulators, and antioxidant enzyme activity of leaves. The study provided a comprehensive analysis of the growth response mechanisms of Limonium species under saline and alkaline stress. The findings improve the understanding of seed germination and salt tolerance under saline and alkaline conditions, deepen the knowledge of the physiological mechanisms of salt tolerance in Limonium species, and will enrich the research on halophyte ecology and seed biology. Additionally, this study provided theoretical value for ecological restoration of saline–alkaline soils and the development and utilization of horticultural plant resources. The results are of significant importance for the efficient use of saline–alkaline land resources and regional ecological environmental improvement.

2. Materials and Methods

2.1. Plant Materials

The plant materials used in this study were seeds of Limonium aureum, Limonium bicolor, and Limonium gmelinii, which were collected in the autumn of 2022 from the Minqin Desert Botanical Garden in China (103.011846° E, 38.718570° N). For each species, seeds were randomly collected from multiple mature, phenotypically healthy, and spatially dispersed mother plants within their ex situ conservation populations. These seeds were then thoroughly mixed to form composite seed batches representing the within-population variation in each species. After collection, the seeds were air-dried naturally in a well-ventilated indoor environment. Subsequently, the air-screen cleaning method was employed to remove impurities and accessory structures (such as withered calyces) [33]. Following cleaning, immature, damaged, insect-bored, or pathogen-contaminated seeds were discarded, and only intact, healthy, and mature seeds were retained for the experiments. Finally, the seed samples were stored at 4 °C in the dark and under sealed conditions until use.

2.2. Seed Morphometric Analysis

The three Limonium species exhibited eight seed phenotypic traits, namely thousand-grain weight (TGW, g), seed length (SL, mm), seed width (SW, mm), seed thickness (ST, mm), seed length-to-width ratio (SLR), shape index (I), seed area (SA, cm²) and seed perimeter (SP, cm).

The TGW was measured using an electronic balance (PTX-FA210S, Huazhi (Fujian) Electronic Technology Co., Ltd., Putian, China) with an accuracy of 0.01 g. The SL, SW, and ST were measured with a vernier caliper (150 mm, DEGUQMNT, Shanghai, China) with an accuracy of 0.01 cm. SLR, defined as the ratio of SL to SW, is used to evaluate the seed aspect ratio. The index I quantifies the deviation of seed shape from a perfect circle. Both indices were calculated from the measured traits. SA and SP were calculated using ImageJ v1.54 (National Institutes of Health, Bethesda, MD, USA). The calculation formula for the shape index (I) was as follows [34]:

$$I=3({SL}^2+{SW}^2+{ST}^2)-{(ST+SW+ST)}^2/3^2$$

(1)

I represents the seed shape index. SL, SW, and ST are the transformed values of the seed’s length, width, and thickness, respectively, which are converted before calculating I. This method sets the seed length as 1, and then calculates the ratios of seed width and height to seed length. For perfectly spherical seeds, the minimum value of I is 0, while for needle-shaped or disk-shaped seeds, the maximum value of I is approximately 0.3.

2.3. Salt and Alkali Stress Experiment

Healthy and uniform seeds were surface-sterilized by soaking in 2% sodium hypochlorite solution (Chengdu Kelong Chemicals Co., Ltd., Chengdu, China) for 10–15 min, followed by three rinses with distilled water. Surface moisture was removed using sterile filter paper (Whatman, Maidstone, UK). Based on preliminary trials, salt stress was imposed with sodium chloride (NaCl, Tianjin Guangfu Technology Development Co., Ltd., Tianjin, China) at concentrations of 100, 200, and 300 mM, and alkali stress with sodium bicarbonate (NaHCO₃, Tianjin Beichen Fangzheng Reagent Factory, Tianjin, China) at the same concentrations. Distilled water served as the control (CK). Seed germination assay was conducted using the Petri dish filter paper method. Briefly, 50 seeds were evenly placed on double-layered sterilized filter papers in 9 cm diameter Petri dishes, with three biological replicates per treatment. For experimental groups, 8 mL of the different concentrations of NaCl and NaHCO₃ solutions was used to moisten the filter papers, while distilled water was used for the control group (CK). All dishes were sealed with Parafilm (Bemis Company, Inc., Neenah, WI, USA) to prevent water loss and minimize evaporation and external contamination, and then incubated in a growth chamber at 25 ± 1 °C under complete darkness. The filter paper and treatment solution in Petri dishes were replaced daily to maintain stable salt concentrations. Germination was defined as radicle emergence exceeding 1 mm in length. Germinated seeds were counted every 24 h until no further germination occurred for three consecutive days.

The physiological and biochemical responses of seedlings to salt and alkali stress were evaluated using a hydroponic system. Three-month-old seedlings at a uniform vegetative growth stage, characterized by approximately 7–8 fully expanded true leaves and no bolting, were selected. These seedlings were originally grown in soil-filled pots (10 seedlings per pot), carefully uprooted, and their roots gently rinsed to remove residual soil before being transferred to the hydroponic system. They were then exposed to Hoagland nutrient solution (Hangzhou Best Biotechnology Co., Ltd., Hangzhou, China) supplemented with 0, 100, 200, or 300 mM NaCl or NaHCO₃ for 24 h, with the 0 mM treatment serving as the control. For each treatment, three independent biological replicates were established, each corresponding to one original pot of 10 seedlings placed together in a single hydroponic container. The 10 seedlings from the same original pot were harvested together and pooled as a composite sample for biochemical assays. All assays included at least two technical replicates. After the treatment was completed, the seedlings were harvested, and the above ground parts (leaves) and underground parts (roots) of the plants were separated. The leaf samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C for subsequent determination of antioxidant enzyme activities and osmolyte contents.

2.4. Indicators of Responses to Salt and Alkali Stress

Seed germination parameters including germination rate (GR), germination potential (GP), germination index (GI), vigor index (VI), and salt tolerance index (STI) were calculated according to established methods.

Germination Rate (GR): The percentage of normally germinated seeds at each time point, calculated as:

$$GR (\%)={\displaystyle \frac{Total number of normally germinated seeds}{Number of seeds tested}}\times 100$$

(2)

Germination Potential (GP): The maximum germination percentage reached during the experiment, calculated as:

$$GP \left(\%\right) ={\displaystyle \frac{Number of normally germinated seeds \left(when the daily germinated seeds reached maximum\right)}{Number of seeds tested}}\times 100$$

(3)

Germination Index (GI): A measure of germination speed and uniformity, calculated as:

$$GI=\sum Gt/Dt$$

(4)

where Gt is the number of germinations at time t, and Dt is the corresponding germination days.

Vigor Index (VI): An integrated indicator of germination vigor, calculated as [29]:

$$VI=GI\times radicle length$$

(5)

Radicle length was measured with a ruler as the distance from the seed base to the radicle tip.

Salt Tolerance Index (STI): A relative measure of growth performance under stress, calculated as [35]:

$$STI (\%)={\displaystyle \frac{Total Growth under salt stress}{Total Growth under control}}\times 100$$

(6)

At the end of the stress treatment, a subset of plants from each group was randomly selected for biomass determination. The plants were first rinsed with tap water, and then separated into roots and leaves. After blotting dry with absorbent paper, the fresh weights of roots and leaves (RFW, LFW) were recorded immediately. The samples were then placed in kraft paper bags, heated in an oven at 110 °C for 5 min, and subsequently dried at 80 °C to a constant weight. The dry weights of roots and leaves (RDW, LDW) were recorded, and the total biomass per unit area (TB) was calculated by summing the dry weight of shoots and roots [36].

The concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car) were calculated following the corresponding equations [37]. Total chlorophyll content (Chl a + b) was also determined. Malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) colorimetric method [38]. Soluble protein (SP) content was measured using the Coomassie Brilliant Blue G-250 method [39]. Soluble sugar (SS) content was determined using the anthrone method [40], and proline (Pro) content was determined using the acidic ninhydrin colorimetric method [41]. Superoxide dismutase (SOD) activity was determined via a modified nitroblue tetrazolium (NBT) method [42], with minor modifications to centrifugation parameters (during enzyme extraction) and reagent dosage ratios in the reaction system. Specific steps were as follows: Plant samples were homogenized with a pre-chilled mortar and pestle. The homogenate was centrifuged at 12,000× g for 20 min at 4 °C using a centrifuge (TGL-20M, Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., Shanghai, China), and the supernatant was collected as the enzyme extract. The reaction system was prepared in a test tube by adding sequentially: 0.1 mL enzyme extract, 1.5 mL 50 mM phosphate buffer (pH 7.8), 0.3 mL 130 mM methionine (Met), 0.3 mL 750 µM NBT, 0.3 mL 100 µM EDTA-Na₂, 0.3 mL 20 µM riboflavin, and 0.5 mL distilled water. The illumination conditions for the NBT photoreduction reaction were strictly controlled. After mixing well, the tubes were placed in a custom-built illumination chamber equipped with cool-white fluorescent lamps (positioned 20 cm above the sample rack) and exposed to a defined light intensity of 4000 ± 100 lx for 20 min. The light intensity was calibrated daily at the tube position using a digital lux meter to ensure inter-assay consistency. To confirm the light-dependency of the NBT reduction, a set of control tubes wrapped in aluminum foil (dark control) was included in every assay. Following the illumination, the absorbance of the mixture was measured at 560 nm, and the SOD activity was calculated. Catalase (CAT) activity was measured using a spectrophotometer [43]. Sodium (Na⁺) and potassium (K⁺) ion contents were determined using a flame photometer (FP6410, Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China) [35].

2.5. Data Analysis

Experimental data were processed and analyzed using Excel 2019 (Microsoft Corporation, Redmond, WA, USA) and SPSS 27.0 (IBM Corporation, Armonk, NY, USA). One-way analysis of variance (ANOVA) was performed to assess differences among the three Limonium species under varying salinity and alkali levels, followed by multiple mean comparisons using Tukey’s HSD test. When the assumption of homogeneity of variance was not met, Tamhane’s T2 test was applied for multiple mean comparisons. Differences were considered statistically significant at p ≤ 0.05. The means and standard errors (SE) of each treatment were calculated. Pearson’s correlation analysis was conducted to explore relationships among germination, growth, and biochemical parameters under NaCl and NaHCO₃ stress. Principal component analysis (PCA) was used to explore the relationships between salt stress, alkali stress and leaf biochemical characteristics, respectively. Figures were generated using Origin 2024 (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Seed Phenotypic Traits of the Three Limonium Species

The mean values, standard errors, and multiple comparison results for eight quantitative traits of the three Limonium species (

Table 1. Comparison of Seed Phenotypic Traits Among the Three Limonium Species.

Species	TGW/g	SL/mm	SW/mm	ST/mm	SLR	I	SA/cm2	SP/cm
L. aureum	0.684 ± 0.063 a	2.457 ± 0.116 a	0.777 ± 0.062 a	0.733 ± 0.099 a	3.169 ± 0.154 a	0.107 ± 0.002 a	0.017 ± 0.003 a	0.621 ± 0.044 a
L. bicolor	0.470 ± 0.070 b	1.937 ± 0.108 b	0.674 ± 0.039 a	0.634 ± 0.069 a	2.875 ± 0.091 a	0.098 ± 0.001 b	0.015 ± 0.004 b	0.590 ± 0.066 b
L. gmelinii	0.411 ± 0.021 b	2.103 ± 0.065 b	0.727 ± 0.046 a	0.680 ± 0.111 a	2.902 ± 0.204 a	0.099 ± 0.004 b	0.016 ± 0.007 b	0.530 ± 0.087 b

Note: Values are presented as mean ± SE. Different lowercase letters in the same column indicate significant differences among species at p ≤ 0.05. TGW, thousand-grain weight (g); SL, seed length (mm); SW, seed width (mm); ST, seed thickness (mm); SLR, seed length-to-width ratio; I, shape index; SA, seed area (cm²); SP, seed perimeter (cm).

) showed that there were significant differences among the species (p ≤ 0.05). L. aureum exhibited the highest values in most traits, including TGW (0.684 g), SL (2.457 mm), SW (0.777 mm), ST (0.733 mm), SLR (3.169), I (0.107), SA (0.017 cm²) and SP (0.621 cm). L. bicolor generally showed intermediate or relatively low levels, and among them, SL (1.937 mm), SW (0.674 mm), ST (0.634 mm), SLR (2.875), I (0.098), and SA (0.015 cm²) were the lowest. L. gmelinii had the lowest TGW (0.411 g) and SP (0.530 cm) among the three species, and its SL (2.103 mm) fell between those of L. aureum and L. bicolor. Overall, the seeds of L. aureum were generally larger and fuller, while those of L. bicolor were the smallest with the smallest surface area. In contrast, L. gmelinii seeds had the lightest mass. These morphological differences provided a basis for understanding the variations in seed germination and salt and alkali tolerance across the different species.

3.2. Saline and Alkaline Stress Responses

3.2.1. Effects of Salt Stress and Alkali Stress on Seed Germination of the Three Limonium Species

Under NaCl stress, the GR activity of L. aureum decreased with the increase in concentration. The GR activities of L. bicolor and L. gmelinii were slightly higher than those of the control when the sodium chloride concentration was 100 mM, but there was no significant change. Subsequently, they decreased significantly as the stress concentration increased. Moreover, inter-specific differences did not reach statistical significance (p > 0.05). Under NaHCO₃ stress, the GR activity of all three Limonium species continuously decreased as the concentration increased. When the concentration of NaCl or NaHCO₃ stress reached 200 mM and above, the GR activity of the three Limonium species was significantly lower than that of the control group (Figure 1A,B). In addition, the GP activity of L. aureum was slightly higher than that of the control when the sodium chloride concentration was 100 mM, but the difference was not significant. Subsequently, it showed a significant downward trend as the stress concentration increased continuously. However, the GP activity of L. bicolor and L. gmelinii showed a significant decreasing trend with increasing NaCl concentration, with significant interspecific differences. Under NaHCO₃ stress, the GP activity of the three species continuously decreased as the concentration of NaHCO₃ increased. Significant interspecific differences were observed at 100 mM and 200 mM, and the GP activity of L. aureum was significantly superior to that of the other two species at 300 mM NaCl or NaHCO₃ (Figure 1C,D). Under NaCl stress, the GI of the three Limonium species all decreased with increasing concentration, with significant interspecific differences at 100 mM and 300 mM. Under NaHCO₃ stress, compared with the control group, the GI of L. aureum showed no significant change at 100 mM and then decreased significantly, while the GI of L. bicolor and L. gmelinii showed a continuous decreasing trend with increasing concentration. At 200 mM NaHCO₃, significant interspecific differences were detected among the three species (Figure 1E,F). The variation pattern of VI in the three Limonium species under NaCl stress was highly consistent with that of GP under NaCl stress. Under NaHCO₃ stress, the VI of L. aureum and L. bicolor continuously decreased with increasing concentration. However, for L. gmelinii, the VI increased significantly at 100 mM, and then showed a decreasing trend with the continuous increase of NaHCO₃ concentration. Significant interspecific differences were observed at 100 mM and 300 mM NaHCO₃ treatments, and the VI of L. aureum was significantly higher than that of L. bicolor and L. gmelinii at 300 mM NaCl or NaHCO₃ (Figure 1G,H).

As shown in Figure 2, at a concentration of 100 mM NaCl or NaHCO₃, the STI of the three Limonium species (L. aureum, L. bicolor, and L. gmelinii) was maintained between 0.80 and 1.00, and inter-specific differences did not reach statistical significance (p > 0.05). With the increase in NaCl concentration, the STI continuously decreased. When the NaHCO₃ concentration exceeded 100 mM, the STI showed a significant decreasing trend, accompanied by significant interspecific differences. At 300 mM NaHCO₃, L. gmelinii had the highest STI, followed by L. aureum, while L. bicolor had the lowest STI.

Through the analysis of

Table 2. Fresh Weight, Dry Weight, and Biomass of The Three Limonium Species Under Saline and Alkali Stress. Values represent the mean ± SE (n = 3).

Index	Treatment (mM)	L. aureum	L. bicolor	L. gmelinii
Leaf fresh weight/g	0	3.75 ± 0.06 Aa	2.68 ± 0.24 Ba	4.55 ± 0.16 Aa
	NaCl 100	14.18 ± 0.06 Aa	2.42 ± 0.26 Ba	3.53 ± 0.26 ABb
	NaCl 200	3.24 ± 0.09 Ab	2.24 ± 0.09 Ba	2.68 ± 0.20 ABbc
	NaCl 300	2.33 ± 0.09 Ac	2.05 ± 0.09 Aa	2.37 ± 0.13 Ac
	NaHCO3 100	3.05 ± 0.06 Aa	2.17 ± 0.03 Bc	2.58 ± 0.28 ABab
	NaHCO3 200	2.58 ± 0.10 Aa	2.44 ± 0.09 Ba	2.50 ± 0.16 Aa
	NaHCO3 300	2.46 ± 0.19 Aa	2.05 ± 0.05 Aa	2.33 ± 0.02 Aa
Root fresh weight/g	0	2.87 ± 0.06 Aa	2.54 ± 0.12 Aa	2.30 ± 0.21 Aab
	NaCl 100	2.45 ± 0.30 Aa	2.73 ± 0.30 Aab	2.06 ± 0.11 Aab
	NaCl 200	2.53 ± 0.14 Aa	2.50 ± 0.14 Aab	2.48 ± 0.07 Aa
	NaCl 300	2.60 ± 0.38 Aa	1.68 ± 0.37 Ab	1.79 ± 0.10 Ab
	NaHCO3 100	2.55 ± 0.01 Bb	2.88 ± 0.03 Aa	1.71 ± 0.04 Cc
	NaHCO3 200	2.49 ± 0.04 Ba	3.58 ± 0.17 Ab	2.51 ± 0.04 Bc
	NaHCO3 300	1.60 ± 0.15 Ba	2.31 ± 0.07 ABa	2.57 ± 0.20 Aa
Leaf dry weight/g	0	0.57 ± 0.03 Aa	0.53 ± 0.05 Aa	0.68 ± 0.04 Aa
	NaCl 100	0.53 ± 0.03 Aab	0.32 ± 0.01 Bb	0.59 ± 0.05 Aab
	NaCl 200	0.51 ± 0.03 Aab	0.30 ± 0.01 Abc	0.55 ± 0.14 Aab
	NaCl 300	0.37 ± 0.04 Ab	0.21 ± 0.01 Bc	0.38 ± 0.04 Ab
	NaHCO3 100	0.56 ± 0.05 Aa	0.42 ± 0.02 Aa	0.51 ± 0.06 Aa
	NaHCO3 200	0.37 ± 0.03 Aa	0.37 ± 0.03 Aa	0.34 ± 0.04 Aa
	NaHCO3 300	0.32 ± 0.1 Aa	0.26 ± 0.02 Aa	0.32 ± 0.01 Aa
Root dry weight/g	0	0.11 ± 0.01 Ba	0.09 ± 0.01 Ba	0.18 ± 0.01 Aa
	NaCl 100	0.08 ± 0.01 Ba	0.07 ± 0.01 Ba	0.15 ± 0.02 Aab
	NaCl 200	0.10 ± 0.01 Aa	0.07 ± 0.01 Aa	0.12 ± 0.01 Aab
	NaCl 300	0.09 ± 0.01 ABa	0.06 ± 0.01 Ba	0.10 ± 0.01 Ab
	NaHCO3 100	0.09 ± 0.01 Ab	0.05 ± 0.01 Ba	0.08 ± 0.04 ABc
	NaHCO3 200	0.10 ± 0.01 Aa	0.05 ± 0.01 Aa	0.10 ± 0.01 Aa
	NaHCO3 300	0.09 ± 0.01 Ab	0.06 ± 0.01 Bc	0.10 ± 0.01 Aa
Total biomass/g·m−2	0	0.69 ± 0.04 Aa	0.62 ± 0.06 Aa	0.81 ± 0.10 Aa
	NaCl 100	0.63 ± 0.05 Aab	0.40 ± 0.02 Bb	0.65 ± 0.01 Aab
	NaCl 200	0.61 ± 0.05 Aab	0.36 ± 0.01 Bbc	0.47 ± 0.04 ABb
	NaCl 300	0.46 ± 0.04 Ab	0.27 ± 0.01 Bc	0.49 ± 0.04 Ab
	NaHCO3 100	0.66 ± 0.05 ABab	0.47 ± 0.02 Bc	0.70 ± 0.05 Aa
	NaHCO3 200	0.47 ± 0.36 Aa	0.44 ± 0.02 Aa	0.42 ± 0.01 Aa
	NaHCO3 300	0.50 ± 0.10 Aa	0.34 ± 0.03 Aa	0.40 ± 0.01 Aa

Note: Different uppercase letters denote significant differences between species within the same treatment; different lowercase letters denote significant differences among treatments within the same species (p ≤ 0.05).

, it was found that with the increase in the stress concentrations of NaCl and NaHCO₃, various growth indices of the three Limonium species showed a downward trend to varying degrees. Under the control (CK), the fresh leaf weight, fresh root weight, dry root weight, and total biomass of L. gmelinii reached the highest values, which were 4.55 g, 2.30 g, 0.16 g, and 0.84 g·m⁻² respectively. When the NaCl concentration was 300 mM, the fresh leaf weight, dry leaf weight, dry root weight, and total biomass of L. bicolor were significantly lower than those of the other two Limonium plants, being 2.05 g, 0.21 g, 0.06 g, and 0.27 g·m⁻² respectively. When the NaHCO₃ concentration was 300 mM, the fresh leaf weight, dry leaf weight, dry root weight, and total biomass of L. bicolor reached the lowest values, which were 2.05 g, 0.26 g, 0.06 g, and 0.34 g·m⁻² respectively. The two types of stress led to a decrease in the fresh and dry weights of leaves and roots, as well as the total biomass of the three Limonium species, among which the fresh leaf weight, dry leaf weight, and total biomass decreased significantly.

3.2.2. Effects of Saline and Alkali Stress on Morphological Characteristics of Seedlings

Figure 3 showed that increasing NaCl and NaHCO₃ concentrations induced concentration-dependent morphological damage in the three Limonium species, progressing from slight curling and wrinkling to severe wilting, drooping, and dehydration. Under NaCl stress, leaves remained relatively stable at 100 mM, whereas at 200 mM, curling and drooping intensified, with L. bicolor showing the most pronounced loss of turgor. At 300 mM, L. aureum and L. gmelinii exhibited severe wilting, while L. bicolor leaves nearly collapsed. A similar but more severe pattern occurred under NaHCO₃ stress. At 100 mM, only mild curling was observed, but by 200 mM, L. bicolor showed clear dehydration and drooping. At 300 mM, both L. aureum and L. gmelinii exhibited severe curling and limpness, while L. bicolor leaves almost completely collapsed. Collectively, L. bicolor was highly sensitive to both NaCl and NaHCO₃ stress, whereas L. aureum and L. gmelinii exhibited comparatively greater tolerance.

3.2.3. Dynamic Changes in Na⁺ and K⁺ Contents Under Saline and Alkali Stress

This study analyzed the responses of Na⁺ and K⁺ contents in the leaves of three Limonium species to the stresses of NaCl and NaHCO₃. The results showed that the salt type, concentration, and plant species had highly significant effects on the ion contents. Specifically, the Na⁺ content in the leaves of L. aureum was significantly higher than that of L. bicolor and L. gmelinii under different NaCl concentrations. The Na⁺ content in the leaves of L. bicolor and L. gmelinii increased with the increase in NaCl concentration, reaching the maximum value when the NaCl concentration was 300 mM. There were significant differences among the three Limonium species under different NaCl stresses. In addition, the Na⁺ content in the leaves of L. aureum increased with the increase in NaHCO₃ concentration, peaking when the concentration increased to 300 mM. The highest Na⁺ content in the leaves of L. bicolor and L. gmelinii occurred under the NaHCO₃ stresses of 100 mM and 200 mM, respectively. There were significant inter-species differences under different NaHCO₃ concentrations. The Na⁺ content in the leaves of the three Limonium species was significantly higher than that of the control when the concentrations of NaCl and NaHCO₃ were 100 mM or above (Figure 4A,B). The K⁺ content in the leaves of the three Limonium species changed significantly under different concentrations of NaCl and NaHCO₃ stresses, but their response patterns were significantly different. It is worth noting that when the concentrations of NaCl and NaHCO₃ were 100 mM, the K⁺ content in the leaves of L. bicolor was the highest, increasing by 31.42% and 55.53%, respectively, compared with the control. When the concentrations of NaCl and NaHCO₃ were 200 mM, the K⁺ content in the leaves of L. bicolor was the lowest. However, when the two stress concentrations increased to 300 mM, the K⁺ content in the leaves of L. bicolor increased significantly, and there were significant inter-species differences under different concentrations of NaCl and NaHCO₃ stresses (Figure 4C,D).

By analyzing the responses of Na⁺ and K⁺ ion contents in the roots of L. aureum, L. bicolor, and L. gmelinii to NaCl and NaHCO₃ stresses, it was found that under both NaCl and NaHCO₃ stresses, the Na⁺ content in the roots significantly accumulated with the increase in stress concentration, while the K⁺ content in the roots showed a significant deficit. Moreover, at the same concentration, the stress effect of NaHCO₃ was significantly stronger than that of NaCl (Figure 5). Under different concentrations of NaCl stress, the Na⁺ content in the roots of L. aureum was significantly lower than that of L. bicolor and L. gmelinii, and there were significant inter-species differences at different NaCl concentrations. When the NaHCO₃ concentration reached 300 mM, the Na⁺ content in the roots of L. gmelinii was significantly higher than that of the other two plants, and significant inter-species differences were observed at other NaHCO₃ concentrations except 200 mM. However, under different concentrations of NaCl and NaHCO₃ stresses, the K⁺ content in the roots of L. gmelinii was significantly higher than that of L. bicolor and L. aureum. When the stress concentration was 300 mM, the K⁺ deficit in the roots caused by NaHCO₃ treatment was significantly greater than that by NaCl. Compared with the control, the K⁺ contents in the roots of L. aureum, L. bicolor, and L. gmelinii decreased by 49.07%, 37.51%, and 35.99%, respectively, when the NaHCO₃ concentration was 300 mM. When the NaCl concentration was 300 mM, these three plants decreased by 40.07%, 30.26%, and 1.86%, respectively, compared with the control.

3.2.4. Effects of Saline and Alkali Stress on Photosynthetic Pigment Contents

As shown in Figure 6, the photosynthetic pigments (Chl a, Chl b, Chl a + b, and Car) of the three Limonium species exhibited different response patterns and significant inter-species differences under NaCl and NaHCO₃ stresses. Overall, the content of Chl a was the highest, followed by Chl b, while the content of Car was always the lowest. Under NaCl stress, the contents of Chl a, Chl b, Chl a + b, and Car in L. aureum increased with the increase in NaCl concentration, reaching the maximum value at 200 mM, and slightly decreased at 300 mM but still remained higher than the control. Under NaHCO₃ stress, the contents of various pigments in L. aureum also increased with the increase in concentration and reached the highest value at 300 mM, with significant inter-species differences. L. bicolor and L. gmelinii showed two different response patterns under NaCl stress. The first pattern was reflected in the changes in Chl a and Car contents. Specifically, the contents of Chl a and Car in L. bicolor significantly decreased to the minimum value at 200 mM (except for the Car content of L. gmelinii), and significantly increased at 300 mM but were still lower than the control. The second pattern was manifested in the changes in Chl b and Chl a + b. That is, the contents of Chl b and Chl a + b in L. bicolor reached the highest value at 300 mM, while those in L. gmelinii peaked at 200 mM, slightly decreased at 300 mM but remained higher than the control. When the NaHCO₃ concentration was 200 mM, the contents of Chl a and Chl b in L. bicolor were the highest, while the Car content was the lowest; the Car content in L. gmelinii was the highest. When the NaHCO₃ concentration increased to 300 mM, the contents of Chl a and Car in L. bicolor and the Car content in L. gmelinii increased and were higher than the control. Conversely, the contents of Chl a + b and Car in L. bicolor and the contents of Chl a, Chl b, and Chl a + b in L. gmelinii decreased at a NaHCO₃ concentration of 300 mM, showing obvious inter-species differences.

3.2.5. Response of Membrane Lipid Peroxidation (MDA) to Saline and Alkali Stress

Figure 7 shows the changes in MDA content of the three Limonium species under different concentrations of NaCl and NaHCO₃ stresses. Specifically, with the increase in NaCl concentration, the MDA content in the three Limonium plants showed a continuous upward trend, reaching the maximum value when the concentration reached 300 mM. Compared with the control, the MDA content in L. aureum, L. bicolor, and L. gmelinii increased by 29.47%, 127.68%, and 88.88%, respectively. Conversely, NaHCO₃ stress reduced the accumulation of MDA in the three Limonium species. The MDA content in all three plants reached the minimum value when the NaHCO₃ concentration was 100 mM, decreasing by 52.74%, 26.44%, and 52.63%, respectively, compared with the control. Subsequently, as the NaHCO₃ concentration increased to 300 mM, the MDA content in the three Limonium species increased but remained lower than the control. Under different concentrations of NaCl and NaHCO₃ treatments, inter-specific differences did not reach statistical significance (p > 0.05).

3.2.6. Changes in Osmotic Adjustment Substances in Response to Saline and Alkali Stress

NaCl and NaHCO₃ stresses significantly induced the Pro content in the three Limonium species. Under different concentrations of NaCl stress, the three Limonium species exhibited two different response patterns. The first pattern was observed in L. aureum and L. gmelinii. Compared with the control, the Pro content in these two plants increased with the increase in NaCl concentration. Under the NaCl stress of 300 mM, the Pro accumulation in both plants reached the maximum value. The Pro content in L. gmelinii increased by 96.71% compared with the control, while that in L. aureum had a greater increase, reaching 177.36%. In contrast, L. bicolor presented the second pattern, in which the highest Pro accumulation occurred under the NaCl stress of 100 mM. However, when the NaCl concentration increased to 300 mM, although the Pro content was still higher than that of the control, it began to decline (Figure 8A). The Pro content in the leaves of L. gmelinii was higher than that of the other two plants at the NaHCO₃ concentrations of 100 mM and 200 mM. The maximum Pro content in the leaves of L. aureum appeared when the NaHCO₃ concentration was 300 mM, but it was still the lowest among the three plants at the same concentration. Significant inter-species differences were observed only when the NaCl concentration was 100 mM and the NaHCO₃ concentration was 300 mM (Figure 8B).

NaCl stress increased the SS content in L. aureum, while it decreased in L. bicolor and L. gmelinii. When the NaCl concentration was 300 mM, the SS content in L. aureum increased by 14.04% compared to the control, while that in L. bicolor and L. gmelinii decreased by 34.51% and 48.39%, respectively (Figure 9A). NaHCO₃ stress increased the SS content in L. aureum and L. bicolor, but decreased it in L. gmelinii. When the NaHCO₃ concentration was 300 mM, the SS content in L. aureum and L. bicolor increased by 0.23% and 21.01%, respectively, compared to the control, while that in L. gmelinii decreased by 10%. When the NaHCO₃ concentration reached 300 mM, the SS content in L. aureum was higher than that in the other two plants. Under all three NaHCO₃ stress concentrations, inter-specific differences did not reach statistical significance (p > 0.05), unlike in the control group (Figure 9B).

Both NaCl and NaHCO₃ stresses significantly decreased the SP content in the three Limonium species (Figure 10). When the NaCl concentration exceeded 200 mM, the SP content in L. aureum, L. bicolor, and L. gmelinii declined, reaching the lowest value at 300 mM. Compared with the control, it decreased by 22.86%, 47.52%, and 60.97%, respectively. Similarly, when the NaHCO₃ concentration reached 300 mM, the SP content in the three Limonium species also decreased significantly, by 7.97%, 38.99%, and 24.48%, respectively, compared with the control. At a NaCl or NaHCO₃ concentration of 300 mM, the SP content in L. aureum was significantly higher than that in L. bicolor and L. gmelinii.

We also measured the changes in the activities of SOD and CAT in the leaves of the three Limonium species under salt and alkali stresses. As shown in Figure 11, under NaCl stress, the activities of SOD and CAT in the three Limonium plants first increased, reaching their maximum values at 200 mM, and then decreased when the NaCl concentration increased to 300 mM. The SOD activity of L. aureum was lower than that of the other two plants at all concentrations, while its CAT activity was higher. Under the NaHCO₃ stress at a concentration of 100 mM, L. gmelinii had the highest SOD activity, and L. aureum had the highest CAT activity. However, under the NaHCO₃ stress at 200 mM, L. aureum had the highest SOD activity, and L. gmelinii had the highest CAT activity. When the NaHCO₃ concentration was 300 mM, L. aureum had the highest SOD and CAT activities. In contrast, L. bicolor had the lowest SOD and CAT activities.

3.2.7. Correlation Analysis of NaCl and NaHCO₃ Stresses on Germination, Growth, and Physiological–Biochemical Parameters

To explore the relationships among various indices during the seed germination and seedling growth stages of the three Limonium species under NaCl and NaHCO₃ stresses, we conducted a Pearson’s correlation analysis. The results are shown in Figure 12. During the seed germination stage (Figure 12A), there were extremely significant positive correlations among the germination and vigor indices such as GR, GP, GI, and VI. The germination vigor indices were extremely significantly positively correlated with the leaf fresh weight and leaf dry weight, as well as with the root fresh weight and root dry weight. The STI was extremely significantly positively correlated with the total biomass and the root dry weight, while its correlation with the leaf fresh weight was not significant. During the seedling growth stage (Figure 12B), there were extremely significant positive correlations among the components of leaf photosynthetic pigments (Chl a, Chl b, Chl a + b, Car). The content of leaf photosynthetic pigments was significantly positively correlated with the leaf K⁺ or Na⁺ ratio, while it was significantly negatively correlated with the leaf Na⁺ concentration. The Pro content was extremely significantly positively correlated with the MDA content, and both were extremely significantly positively correlated with the SS and SP contents. The SOD activity was extremely significantly positively correlated with the CAT activity. Moreover, the SOD was extremely significantly positively correlated with the Pro, MDA, SS, and SP contents, while the correlations between CAT and these indices were not significant.

3.2.8. Principal Component Analysis of NaCl and NaHCO₃ Stresses on Biochemical Parameters

After investigating the effects of different concentrations of NaCl and NaHCO₃ on various parameters during the seed germination and seedling growth stages of the three Limonium species, we conducted a principal component analysis (PCA) to comprehensively analyze the multivariate physiological and biochemical responses. To ensure the validity and robustness of the PCA, we optimized the initial variable set prior to the analysis to eliminate the interference of high collinearity on the model structure. Based on the contribution of each index to variance and the reduction in redundancy, 10 key indices under NaCl stress and 9 key indices under NaHCO₃ stress were ultimately retained for conducting the PCA. After standardizing the data, the suitability of factor analysis was evaluated using the Kaiser–Meyer–Olkin (KMO) test and Bartlett’s test of sphericity (

Table 3. Kaiser–Meyer–Olkin and Bartlett’s test of sphericity.

Stress Types	Kaiser–Meyer–Olkin Measure of Sampling Adequacy	Bartlett’s Test of Sphericity
		Approx. Chi-Square	df	Sig.
NaCl	0.615	202.679	55	0.000
NaHCO3	0.604	61.844	36	0.005

). The KMO values were 0.615 for the NaCl treatment and 0.604 for the NaHCO₃ treatment, indicating that the sample data had adequate sampling adequacy for PCA. The results of Bartlett’s test of sphericity showed that the chi—square values were χ² = 202.679 (p < 0.001) for the NaCl treatment and χ² = 61.844 (p = 0.005) for the NaHCO₃ treatment, confirming significant correlations among the retained variables. These results indicated that the selected indices were suitable for principal component analysis.

The PCA results showed that the first four principal components extracted under NaCl stress accounted for a cumulative variance of 77.09%, which effectively reflected most of the trait variation (Figure 13A). Among them, PC1 (31.68%) was strongly positively correlated with Root Na⁺, MDA, and SOD, and negatively correlated with SS, SP, and Chl b. PC2 (23.39%) was significantly positively correlated with Car and Leaf Na⁺, and negatively correlated with Root K⁺ and Chl b. PC3 (12.68%) was positively correlated with Chl a and Chl a + b, and also with CAT. PC4 (9.33%) was positively correlated with Leaf K⁺ and CAT, and negatively correlated with Car (

Table 4. Principal component vectors, eigenvalues, contribution rate and cumulative contribution rate of first four principal components based on tree means for the 10 traits under NaCl treatment.

Traits	Component
	1	2	3	4
Chl b	0.37	−0.289	0.19	−0.016
Car	−0.223	0.307	−0.299	0.236
Leaf Na+	−0.283	0.362	0.333	0.02
Root K+	0.328	−0.46	0.028	0.043
Root Na+	0.332	0.405	0.026	0.132
MDA	0.205	0.404	0.093	0.169
SS	−0.388	−0.168	0.3	0.147
SP	−0.388	−0.327	−0.031	0.214
SOD	0.385	0.078	0.307	0.076
CAT	−0.113	−0.004	0.748	0.095
Eigenvalue	3.486	2.573	1.395	1.027
% of Variance	31.687	23.391	12.680	9.333
Cumulative %	31.687	55.078	67.757	77.090

). Under NaHCO₃ stress, the first four principal components accounted for a cumulative variance of 70.84%, also capturing the main variation (Figure 13B). PC1 (29.17%) was strongly positively correlated with Chl a, Chl a + b, and Pro. PC2 (15.49%) was significantly positively correlated with Car, SS, and SOD. PC3 (13.49%) was positively correlated with SS, SP, and CAT, and negatively correlated with Root Na⁺ and Pro. PC4 (12.69%) was positively correlated with MDA, and negatively correlated with CAT, Root Na⁺, and Leaf Na⁺ (

Table 5. Principal component vectors, eigenvalues, contribution rate and cumulative contribution rate of first four principal components based on tree means for the 9 traits under NaHCO₃ treatment.

Traits	Component
	1	2	3	4
Car	0.366	−0.39	0.344	0.109
Leaf K+	−0.442	0.227	0.348	0.309
Root K+	0.095	−0.686	0.011	0.343
Root Na+	0.485	0.011	−0.057	−0.401
Pro	0.362	0.354	0.256	−0.21
MDA	−0.269	−0.114	0.225	−0.546
SS	0.238	0.374	0.368	0.446
SOD	0.352	0.03	0.215	0.078
CAT	0.207	0.215	−0.678	0.259
Eigenvalue	2.625	1.394	1.214	1.143
% of Variance	29.172	15.492	13.488	12.696
Cumulative %	29.172	44.664	58.151	70.847

).

Based on the principal component analysis, the comprehensive scores of the three Limonium species under different concentrations of NaCl stress were calculated and ranked (

Table 6. The scores and rankings of the three Limonium species under different NaCl stress based on Principal Component Analysis.

Species	NaCl Concentration	Scores of PC1	Scores of PC2	Scores of PC3	Total Scores	Ranking
L. aureum	0	−22.06	−7.329	9.000	−9.490	9
	100	0.243	−0.014	0.377	0.183	4
	200	−16.786	−197.895	10.824	−45.923	11
	300	0.294	−0.015	0.126	0.079	6
L. bicolor	0	−5.706	173.125	−39.681	26.268	2
	100	0.537	0.025	0.070	0.175	5
	200	1.964	151.001	−11.952	61.433	1
	300	2.676	0.052	0.206	0.726	3
L. gmelinii	0	−0.239	−152.521	−21.360	−29.556	10
	100	−12.895	0.053	−0.064	−3.218	7
	200	−0.608	−41.642	−46.996	−47.412	12
	300	−15.658	−0.012	−0.024	−3.928	8

). It was found that under NaCl stress, L. bicolor attained the highest comprehensive score (61.433) at 200 mM, while L. aureum achieved a relatively high score (0.183) at 100 mM. By contrast, L. gmelinii performed poorest under NaCl stress, with a score of −47.412 at 200 mM. For NaHCO₃ stress, L. gmelinii reached the highest comprehensive score (331.288) at 200 mM, and L. aureum also showed good performance at this concentration (0.600). The lowest score was observed in L. bicolor under control conditions (0 mM NaHCO₃), which was −42.351 (Table 6 and

Table 7. The scores and rankings of the three Limonium species under different NaHCO₃ stress based on Principal Component Analysis.

Species	NaHCO3 Concentration	Scores of PC1	Scores of PC2	Scores of PC3	Total Scores	Ranking
L. aureum	0	−20.666	6.755	−8.025	−2.931	10
	100	0.063	0.612	0.132	0.299	4
	200	15.838	5.830	−22.347	0.600	3
	300	0.074	0.069	0.024	0.098	6
L. bicolor	0	−72.373	−50.388	−30.237	−42.351	12
	100	0.041	−0.066	−0.192	−0.048	9
	200	77.118	−46.777	9.040	−32.080	11
	300	−0.007	−0.045	0.056	0.006	8
L. gmelinii	0	975.323	97.498	16.369	289.192	2
	100	0.004	−0.040	0.117	0.033	7
	200	1163.608	90.607	−1.244	331.288	1
	300	0.007	−0.025	0.723	0.163	5

).

4. Discussion

This study has achieved three main findings, which are of great significance for the current understanding of the stress-adaptation mechanisms of halophytes and for guiding ecological restoration. First, this study integrated the seed phenotypes of three Limonium species with stress conditions. It was discovered that phenotypic indicators such as seed area and thickness are closely related to germination vitality. This makes seed phenotype a simple and reliable stress-resistance indicator, which can be used for the screening of germplasm resources in ecological restoration. Second, research on physiological mechanisms demonstrated that alkaline salts (NaHCO₃) disrupt potassium ion homeostasis and the stability of photosynthetic pigments more severely than neutral salts (NaCl) at the same concentration, revealing their unique physiological toxicity. Finally, through a comprehensive comparison of the response differences in the three plants to NaCl and NaHCO₃, this study elucidated their adaptive strategies based on habitat differences, thus providing a direct basis for the application of specific species in different saline–alkali soil types. In summary, these findings emphasize that in future research, it is essential to distinguish and evaluate alkalinity and salinity as independent abiotic stress factors.

Seed germination is a critical stage in the plant life cycle, especially in saline–alkali environments. The phenotypic characteristics of seeds and their stress-responsive germination behaviors directly determine the species’ regeneration potential and geographical distribution pattern [44]. In this study, we systematically compared the seed morphology and germination characteristics of the three Limonium species under different concentrations of NaCl and NaHCO₃ stresses. The results showed that there were distinct differences in the resistance of the three Limonium species, and the seed phenotypes and stress-response strategies jointly determined this differentiation pattern. Seed morphological traits are pre-adaptation characteristics formed through long-term natural selection. Their variations directly affect the water-absorption efficiency, nutrient storage capacity, and gas exchange efficiency during germination under stress [45]. L. aureum has evolved core phenotypic characteristics such as SA and ST that are tailored to its survival strategy, thus achieving a synergistic balance between diffusion potential and resource reserve. These characteristics facilitate rapid water absorption by the seeds and efficient O₂/CO₂ exchange, and research has confirmed their importance in initiating metabolic activities during the initial stage of germination in hyperosmotic environments [46]. On the other hand, L. aureum is commonly found in inland mixed saline–alkaline environments, which are characterized by a high pH value (pH 8.0–8.8) and a high content of NaHCO₃/Na₂CO₃. The local standard for this species clearly records the characteristics of its habitat, indicating that L. aureum is naturally adapted to saline–alkaline soils with a pH range of 7.5 to 9.0 and a salt content of ≤4%. This is also a typical feature of the alkaline habitats in the inland regions of northwestern China [47]. In contrast, although the overall phenotypic advantages of the other two Limonium species are not as prominent as those of L. aureum, they still possess a certain degree of dispersal ability. However, their limited endosperm reserves are insufficient to support the energy-consuming stress resistant metabolism during germination, which inherently restricts their adaptability [44]. Numerous studies have shown that NaCl and NaHCO₃ stresses inhibit seed germination through different mechanisms. The osmotic stress induced by NaCl stress reduces the water potential of the medium, impeding seed water absorption. Meanwhile, excessive Na⁺ causes ion toxicity, disrupting the intracellular ion balance and the integrity of the membrane system, thus inhibiting seed germination. In contrast, NaHCO₃ stress involves alkaline salts such as HCO₃⁻ and CO₃²⁻. These salts increase the pH of the solution, causing the seed germination process to be affected by osmotic imbalance resulting from high pH, further suppressing germination [31,48]. When the NaCl stress concentration was 300 mM, the GR of L. aureum was higher than that of L. gmelinii, while the GI of L. gmelinii was higher than that of L. aureum. Under the same concentration of NaHCO₃ stress, the GR of L. gmelinii was higher than that of L. aureum, while the GI of L. aureum was higher than that of L. gmelinii. L. aureum is native to slightly saline–alkali soils in semi-arid grasslands. The selective pressures of intermittent NaCl stress and water fluctuations have led to the evolution of a resource-allocation pattern of “antioxidation priority”. Under high-concentration NaCl stress, a higher GR can rapidly scavenge reactive oxygen species (ROS) to resist short-term high-intensity stress, while sacrificing part of the GI to conserve metabolic resources. This rapid response strategy is not unique to Limonium; it has been observed in the halophyte quinoa (Chenopodium quinoa), where rapid antioxidant activation is key to withstanding transient salinity shocks in its native high-altitude habitats [49]. L. gmelinii is naturally distributed in the salinized areas of northern Xinjiang. It grows sympatrically with euhalophytes such as Salicornia europaea and Kalidium foliatum. The soil in its habitat is “saline soil dominated by chlorides”, and the pH of such soil in the desert areas of Xinjiang is generally 8.0–9.5. Meanwhile, seed germination experiments have confirmed that it can still germinate normally under the stress of 200 mM NaCl (corresponding to a soil electrical conductivity (EC) of approximately 8–10 dS/m). Therefore, it can be speculated that its pH adaptation range is most likely between 8.0 and 9.5 [50]. The stable high-salt (especially NaHCO₃) environment has enabled it to develop a stress-specific regulation pattern for defense and growth. Under NaHCO₃ stress, it up-regulates GR to cope with the oxidative damage exacerbated by high pH, while under NaCl stress, it prioritizes maintaining a high GI to adapt to the stable resource supply in wetlands. This strategy results in the highest STI of L. gmelinii under 300 mM salt stress [51]. The constant, high ionic strength in its habitat may have selected for constitutive or rapidly inducible ion homeostasis and osmoprotectant synthesis, allowing it to maintain germination pace even under high NaCl, while the specific challenge of high pH in NaHCO₃ stress requires a boosted antioxidant response. Such a “dual-strategy” adaptation is also evident in the highly alkali-tolerant forage legume, alfalfa (Medicago sativa), which maintains growth under mixed salt-alkali stress by differentially regulating ion transport and antioxidant pathways [52]. Another possible reason is that the thick seed coat of L. aureum seeds may impede water penetration. Meanwhile, Na⁺ can disrupt the enzyme systems involved in nutrient decomposition (such as amylase and protease), and high pH can also disrupt the conversion of starch in the endosperm to soluble sugars, leading to an interruption of energy supply, inhibited radicle elongation, and ultimately blocking the initiation of germination [53,54]. Under both stress conditions, the GP and VI of L. aureum were significantly better than those of the other two species, indicating its overall ability to maintain good physiological activities under adverse conditions. The STI generally decreased with the increase in stress concentration. L. gmelinii had the highest STI at 300 mM, while L. bicolor had the lowest. This may suggest that although the initial adaptability of L. gmelinii is not as good as that of L. aureum, its tolerance mechanism is more robust under high stress. L. bicolor may be less adapted to saline–alkali conditions in its native environment. L. bicolor is native to slightly saline–alkali sandy habitats. L. bicolor is a typical salt-secreting halophyte, which is naturally distributed in coastal areas (such as the Yellow River Delta) and inland saline–alkaline lands. Although there is currently a lack of direct measured data on the soil pH of its native habitat, previous studies have simulated its halohabitat by setting up NaCl stress gradients (0–300 mM), with the simulated soil pH values ranging from 7.0 to 8.5. Moreover, this species shows the best growth state under the 100 mM NaCl treatment (corresponding to a soil pH of 7.5–8.0), with peak values in biomass accumulation, photosynthetic efficiency, and antioxidant enzyme activity [55,56,57]. This result can serve as a strong indirect reference for the suitable pH range of its native habitat. The lack of continuous high-salt selection has led to the lowest GP, VI, and STI. However, it can still maintain basic germination activity through limited metabolic regulation, demonstrating a moderate stress resistance level consistent with the findings of coastal Limonium species [58]. NaHCO₃ stress creates a more complex inhibitory effect through the synergistic action of high pH, osmotic stress, and HCO₃⁻ toxicity. L. bicolor maintains moderate-level germination indices (GI, GP, and vigor index VI) under alkali stress, probably relying on the limited accumulation of osmotic-regulating substances (such as Pro) to alleviate high-pH damage. This is a common adaptation strategy in moderately stress-resistant plants, consistent with previous research that limited proline accumulation can maintain a moderate germination level [59]. Overall, L. aureum shows strong adaptability under NaCl stress, mainly due to its larger SA and ST, which enhance water absorption and gas exchange. L. gmelinii, on the other hand, exhibits stronger alkaline adaptability under NaHCO₃ stress and can adapt to diverse saline–alkali habitats (such as inland soda-saline soils and coastal alkali flats), thus supporting its wide geographical distribution. Although L. bicolor is somewhat restricted during germination, it can still maintain a certain level of germination activity under moderate stress through limited metabolic regulation. These differences reflect the different adaptation strategies of the three Limonium species under different stress conditions, which are closely related to the ecological characteristics of their native environments [60]. These findings suggest that seed morphological traits are not merely physical characteristics but active regulatory factors in stress adaptation. They also highlight that habitat characteristics determine the direction of plant stress-resistance evolution, and plants provide crucial support for their continuous survival in the environment through targeted adaptation strategies, achieving a dynamic fit between the environment and the plants.

Under NaCl and NaHCO₃ stress conditions, the LFW, LDW, RDW, and TB of L. aureum, L. bicolor, and L. gmelinii decreased significantly with increasing concentrations (Table 3), indicating that both salt and alkali stresses inhibited plant growth and biomass accumulation by inducing osmotic stress and ion toxicity, thereby disrupting water absorption and cellular homeostasis, Similar phenomena have also been observed in tomato and Viola tricolor L [61,62,63]. Although the biomass of the three plants generally showed a downward trend, the extent of the decrease varied significantly, reflecting differences in the resistance of the three Limonium species. Under control conditions, L. gmelinii had the highest LFW, RFW, RDW, and TB, demonstrating the strongest growth potential. Under low-and-high concentration NaCl treatments, the LDW, RDW, and TB of L. gmelinii were higher than those of L. aureum. Under low-concentration NaHCO₃ treatments, the total biomass of L. gmelinii was higher than that of L. aureum, while at medium- and high-concentrations (NaHCO₃ ≥ 200 mM), the fresh leaf weight, dry leaf weight, and total biomass of L. aureum were higher than those of L. gmelinii. In contrast, under high-concentration (300 mM) NaCl and NaHCO₃ treatments, L. bicolor had the lowest LFW, LDW, RDW, and TB, further demonstrating that L. aureum and L. gmelinii are more adaptable to saline–alkali environments than L. bicolor. Specifically, L. aureum was more significantly inhibited under NaCl stress, while L. gmelinii and L. bicolor were still more sensitive to NaHCO₃ stress. This may be because NaCl mainly causes ionic stress and osmotic stress, while NaHCO₃, in addition to ionic and osmotic stresses, also superimposes high-pH stress and HCO₃⁻-specific toxicity, forming a more complex synergistic stress effect, resulting in a significantly stronger inhibitory effect on plants than NaCl. This is also consistent with the mainstream mechanism research in the field of saline–alkali stress [64,65]. Furthermore, pre-exposure to mild salinity (e.g., via NaCl or CaCl₂ priming) has been shown to induce cross-tolerance to subsequent water deficit in periwinkle, a phenomenon likely rooted in overlapping osmotic adjustment and antioxidant defense pathways [66]. Given the shared osmotic component between salinity, alkalinity, and drought, it is plausible that the superior performance of L. aureum under high NaHCO₃ stress may partly stem from an inherent capacity for cross-protection across abiotic stress types, warranting targeted investigation in future studies [67]. The additional high-pH stress under alkali conditions is particularly damaging, potentially interfering with plasma membrane H⁺-ATPase activity, disrupting proton gradients essential for nutrient uptake and ion transport, and even affecting cell wall structure and integrity [67]. This study revealed an interesting pattern regarding the inhibitory effects of NaHCO₃ and NaCl on plant growth and biomass accumulation. Specifically, at medium- and low-concentrations (≤200 mM), the inhibitory impact of NaHCO₃ stress was generally more pronounced than that of NaCl. However, when the concentration reached a high level (300 mM), this trend became species—dependent. To understand the underlying mechanism, findings from studies on common bean (Phaseolus vulgaris L.) provide valuable insights. In these studies, it was demonstrated that medium to low concentrations of NaHCO₃ could severely disrupt cellular metabolic homeostasis and ion absorption (such as significantly reducing K⁺ and Ca²⁺ levels) via high-pH-induced effects. Consequently, this disruption directly inhibits the energy source for growth [68].

Salt stress and alkali stress are the main abiotic stress factors that disrupt plant ion balance and osmotic stability. This study found that under NaCl and NaHCO₃ stresses, there were significant differences in the accumulation patterns of Na⁺ and K⁺ in the leaves and roots of L. aureum, L. bicolor, and L. gmelinii. The magnitude and trend of changes varied depending on the species and stress type, reflecting species specific ion regulation mechanisms (Figure 4 and Figure 5). Overall, although both stresses led to an increase in Na⁺ content in the leaves and roots, the effects of the two stresses on the change in K⁺ content showed different trends. However, on the whole, the increase in Na⁺ content and the decrease in K⁺ content presented a typical “high Na⁺-low K⁺” pattern, which is consistent with the ion toxicity mechanism of halophytes [64]. The regulation of ion homeostasis under salt and alkali stress in these species is driven by key mechanisms, such as Na⁺/H⁺ antiporters (e.g., NHX transporters), SOS pathways, and ROS scavenging systems, all of which are well-documented in halophytes. Ion transport proteins such as the NHX exchangers play a crucial role in sequestering Na⁺ into vacuoles, maintaining cytosolic ion balance, and minimizing toxicity in leaves and roots [69]. In addition, high-pH stress exacerbates the need for cell-wall remodeling, particularly in terms of proton gradients, which is vital for nutrient uptake and maintaining turgor pressure in alkaline conditions [70]. Under NaCl stress, L. aureum had the highest Na⁺ content in the leaves but the lowest in the roots, indicating its weak sodium exclusion and ion compartmentalization abilities and relatively weak salt tolerance. The Na⁺ and K⁺ contents in the leaves and roots of L. bicolor were relatively stable, which may rely on its salt-gland secretion function, and its salt-secreting adaptability is moderate. L. gmelinii maintained a low Na⁺ content and a high K⁺ content in both the leaves and roots, and its K⁺/Na⁺ ratio was significantly higher than that of the other two plants. This may be achieved by enhancing the activity of Na⁺/H⁺ antiporters (NHX type) in the roots and plasma membrane H⁺-ATPase, combined with the root ion-compartmentalization mechanism, to reduce the translocation of Na⁺ to the above-ground parts, thus maintaining the proton gradient and ion homeostasis, presenting a typical ion regulation pattern [71]. One important finding of this study lies in the differences in ion accumulation. Under NaHCO₃ stress, the Na⁺ content in the roots of L. gmelinii was significantly higher than that of L. aureum and L. bicolor under the same-intensity NaCl stress. This result suggests that alkaline stress can more effectively induce the absorption and enrichment of Na⁺ in roots compared to neutral salt stress. This specific enrichment of Na⁺ in a high-pH environment not only exacerbates the imbalance of the Na⁺/K⁺ ratio but also increases the degree of ionic stress in root tissues, thus further confirming the common view in the academic community that alkaline salt stress disrupts plant ion homeostasis more severely than neutral salt stress, especially at the level of root ion regulation [72]. Mechanistically, the combined effect of high-concentration HCO₃⁻ and high pH may be the key. Studies on cucumber (Cucumis sativus) have shown that this combined stress specifically inhibits K⁺ absorption and simultaneously promotes Na⁺ accumulation, ultimately exacerbating the ionic toxicity effect in the rhizosphere [72]. In conclusion, when these three plants respond to salt stress and alkali stress, they adopt different ion-regulation and salt-secretion strategies, showing their respective adaptation characteristics.

Salt and alkali stresses can significantly impact plant photosynthesis, mainly by disrupting the synthesis of chlorophyll, which is a key pigment for converting light energy into organic compounds [73]. Chlorophyll content can reflect the intensity of photosynthesis under salt stress and is an important physiological indicator for measuring plant stress resistance [74]. Under different salt and alkali stress conditions, the contents of Chl a, Chl b, and Car in the three Limonium species first increased and then decreased (Figure 6A,H). This phenomenon may be related to the increased production of osmotic adjustment substances in plants under moderate salt and alkali stresses. These substances are direct or indirect by products of photosynthesis [75]. Specifically, L. aureum showed significant pigment accumulation under both stresses. Especially when the NaHCO₃ concentration was 300 mM, the contents of photosynthetic pigments were higher than those under the same concentration of NaCl stress (Figure 6A,H). This phenomenon might be ascribed not merely to the plant’s inherent physiological and biochemical characteristics but also to the specific root-associated microbiome, particularly the endophytic community. A growing body of research has demonstrated that plant endophytes serve as vital symbionts, facilitating the host plants in coping with environmental stresses. In the case of salt-tolerant endophytes, studies on salt-tolerant maize lines have shown they can enhance plant salt tolerance through various direct and indirect mechanisms. For example, these endophytes can produce ACC deaminase, thereby reducing stress—induced ethylene levels. They are also capable of synthesizing plant hormones like indole-3-acetic acid (IAA) to promote root growth. Additionally, they can induce systemic resistance, which activates the plant’s antioxidant system, as evidenced by increased activities of superoxide dismutase (SOD) and catalase (CAT). Moreover, they assist in ion compartmentalization, such as by regulating NHX transporters, to maintain ion homeostasis [76]. Among the three Limonium species, L. gmelinii exhibited relatively higher photosynthetic pigment retention under NaCl stress, suggesting better tolerance to neutral salinity. However, it was particularly sensitive to NaHCO₃ stress, with the most pronounced decline in Chl a and total chlorophyll at high concentrations, indicating a limited capacity to maintain photosynthetic integrity under high-pH saline conditions (Figure 6A,B), indicating that severe stress had seriously inhibited its photosynthetic activity and accelerated pigment degradation. This is completely consistent with the reported finding that high-concentration saline–alkaline stress leads to a sharp loss of chlorophyll [12]. In summary, under different salt and alkali stress conditions, the contents of photosynthetic pigments in the three Limonium species showed significant changes. Especially under NaHCO₃ stress, the photosynthesis of L. gmelinii was significantly inhibited. This indicates that when plants respond to different types of saline–alkaline stresses, the impact on photosynthesis and the differences in their metabolic regulation mechanisms are directly related to their stress resistance performance.

MDA, the end-product of lipid peroxidation, is often used as an important indicator of oxidative stress in plants. An increase in its content usually implies damage to the membrane structure and may induce programmed cell death [73]. The results of this study showed that the MDA content in the leaves of the three Limonium species increased continuously with the increase in NaCl concentration, and the MDA content under high NaCl concentration was higher than that of the control (Figure 7A). The MDA accumulation induced by NaCl stress can be attributed to the disruption of the balance between the production and scavenging of intracellular reactive oxygen species (ROS) [77], thus reducing the cell’s ability to scavenge ROS and accelerating the damage to the membrane system. An unusual phenomenon was observed under NaHCO₃ stress: MDA content in the leaves of all three Limonium species declined at 100 mM and remained below control levels even at higher concentrations (Figure 7B), contradicting the typical expectation that oxidative damage increases with stress intensity. This may be explained by the activation of antioxidant defenses under moderate alkaline stress. Specifically, enzymes such as SOD and CAT are likely upregulated, scavenging ROS more efficiently than they are produced, thereby reducing net lipid peroxidation and MDA accumulation [78]. Furthermore, the high pH associated with NaHCO₃ stress may trigger specific signaling pathways—such as the upregulation of Na⁺/H⁺ antiporters (NHX) and H⁺-ATPases—that help maintain cellular ion homeostasis and mitigate oxidative damage [79]. In addition, the different patterns of MDA changes under different stress types further reveal the differences in membrane damage degree and the efficiency of the antioxidant defense system among different treatments. Under NaCl stress, L. bicolor had the highest MDA content, indicating the strongest oxidative damage; there was no significant difference in MDA levels between L. aureum and L. gmelinii, suggesting that the degrees of oxidative damage to the two were similar. In contrast, under NaHCO₃ stress, the MDA contents of the three plants were relatively low. This phenomenon may be attributed to several factors. Firstly, these plants can maintain pH homeostasis through rhizosphere organic acid secretion and intracellular proton pump regulation. They are also capable of converting HCO₃⁻ into a supplementary carbon source, which enhances the activities of antioxidant enzymes such as superoxide SOD and CAT, thereby inhibiting membrane lipid peroxidation. Secondly, during the determination by the thiobarbituric acid (TBA) colorimetric method, HCO₃⁻ may cause measurement artifacts, leading to biased results. Furthermore, the activation of the abscisic acid (ABA) and jasmonic acid (JA) signaling pathways in the plants, along with the accumulation of osmoregulatory substances such as proline and soluble sugars, synergistically alleviates oxidative damage [80].

Pro, as an osmotic regulator in plants, serves as an indicator of the degree of salt and alkali stress. It can adjust the osmotic potential, scavenge free radicals, and stabilize subcellular structures. It functions not only in osmoregulation but also as a molecular chaperone, a hydroxyl radical scavenger, and a redox buffer, thereby protecting cellular structures and enzymes [81]. Previous studies have shown that proline accumulation is positively correlated with NaCl concentration [82]. In this study, the Pro content in the leaves of the three Limonium species increased with increasing NaCl concentrations. Under high NaCl stress, the Pro content in L. aureum and L. gmelinii significantly increased (Figure 8A). These results suggest that, compared to L. bicolor, L. aureum and L. gmelinii are better able to cope with salt-induced damage by accumulating more Pro, thereby enhancing their adaptability to NaCl stress environments. Interestingly, at 200 mM, all three species accumulated more Pro under NaHCO₃ stress than under an equivalent NaCl concentration (Figure 8B). This indicates a stronger need for osmotic adjustment and redox buffering under alkaline conditions. This phenomenon can be attributed to the cumulative effects of high pH and bicarbonate toxicity, which exacerbate ion imbalance and oxidative stress beyond the levels induced by neutral salts. In these conditions, plants activate the proline biosynthesis pathway (via P5CS), as well as the accumulation of soluble sugars and the upregulation of antioxidant enzymes, to maintain cellular osmotic potential and redox homeostasis, which has been reported in previous studies [83,84,85]. Moreover, NaHCO₃ stress induces more proline accumulation and antioxidant enzyme activation than equivalent NaCl stress, reflecting the more severe physiological challenges posed by alkaline conditions.

Under the stresses of high-concentration NaCl and NaHCO₃, the contents of SS and SP in the leaves of L. gmelinii decreased with the increase in stress concentration, and were generally lower than those of the control (Figure 9 and Figure 10). This indicates that L. gmelinii may actively accumulate osmotic adjustment substances such as SS and SP to regulate the cell osmotic potential, thereby enhancing the cell’s water absorption and water retention abilities. It is worth noting that under NaHCO₃ stress, its SS and SP contents were higher than those under NaCl stress, suggesting that L. gmelinii has a higher tolerance to NaHCO₃ stress than to NaCl stress. Specifically, the SS content in the leaves of L. aureum first increased and then decreased as the NaCl concentration increased, but was still generally higher than that of the control (Figure 9A). This trend is closely associated with the multifunctional roles of soluble sugars (SS) as classic compatible solutes: they accumulate to stabilize biological membranes, protein structures and organelles, balance osmotic potential to prevent cellular dehydration, and act as carbon and energy reserves for post-stress metabolic recovery. The subsequent decline under extreme stress may reflect the depletion of metabolic resources due to excessive energy consumption for stress defense or a shift in resource partitioning toward other prioritized stress-responsive pathways [86]. However, the SP content in the leaves of L. aureum decreased with the increase in NaCl concentration, which may be related to the decrease in photosynthetic activity. This observation is consistent with previous studies, indicating that high salinity can damage plant photosynthesis and protein synthesis. For example, a study on Carthamus tinctorius L. under NaCl and NaHCO₃ stresses found that both types of stresses led to a decrease in chlorophyll content and photosynthetic efficiency, with the effect of NaHCO₃ being more significant [87]. Under the stresses of high-concentration NaCl and NaHCO₃, the SS and SP contents in the leaves of L. aureum were higher than those in L. bicolor and L. gmelinii. This shows that compared with the other two plants, L. aureum exhibits a stronger osmotic adjustment ability under salt and alkali stresses. Under low—concentration NaHCO₃ stress, the SS content in the leaves of L. bicolor increased significantly and was still higher than that of the control at higher concentrations, demonstrating a strong osmotic adjustment ability. Similar phenomena have also been reported in other plants, that is, plants maintain osmotic balance and alleviate oxidative damage by accumulating osmotic adjustment substances (such as sugars and proline) and up-regulating the activities of antioxidant enzymes (such as SOD and CAT) [64].

Under normal conditions, the production and clearance of reactive oxygen species (ROS) in plants are in dynamic equilibrium [88,89]. However, salt and alkali stresses disrupt this balance, leading to excessive ROS production and accumulation, resulting in increased levels of MDA, O₂^•−, and H₂O₂ in seedlings, which ultimately affect plant growth and development [90,91]. In our study, we observed a significant increase in the activities of SOD and CAT in L. aureum, L. bicolor, and L. gmelinii under low and medium NaCl concentrations (Figure 11A,C). These results suggest that the three Limonium species exhibit some degree of adaptation to low and medium NaCl stress. However, under 300 mM NaCl stress, only L. aureum showed a decrease in CAT activity compared to the control (Figure 11C), which may be due to excessive ROS production induced by NaCl stress, impairing antioxidant enzyme activity and cellular function [92]. Under NaHCO₃ stress, SOD and POD activities followed similar trends as under NaCl stress. Significantly, at high NaHCO₃ (300 mM), SOD and CAT activities in all three species’ leaves exceeded those of the control (Figure 11B,D). This sustained upregulation suggests that alkaline stress triggers a systemic retrograde signaling cascade from chloroplasts and mitochondria, involving H₂O₂ and Ca²⁺ waves, which activate mitogen-activated protein kinases (MAPKs) and ultimately induce antioxidant gene expression [93]. Unlike NaCl, which primarily induces osmotic shock, NaHCO₃’s dual challenge of high pH and HCO₃⁻ toxicity necessitates a more persistent and integrated ROS detoxification response. This suggests that the enhanced antioxidant system under NaHCO₃ stress is more effective in scavenging ROS [94]. These results indicate that plants exposed to alkaline conditions possess stronger antioxidant defenses. Compared to NaCl stress, NaHCO₃ stress triggers more persistent activation of antioxidant enzymes, enabling the plants to effectively detoxify ROS and maintain redox homeostasis [95].

Principal component analysis (PCA) based on optimized physiological and biochemical indicators (10 under NaCl stress, 9 under NaHCO₃ stress) revealed significant differences in the tolerance strategies of the three Limonium species to salt and alkali stresses, closely related to their native habitat adaptability. L. bicolor exhibited the highest overall score under NaCl stress, reflecting the strongest tolerance, primarily driven by principal components related to ion homeostasis (e.g., root Na⁺/K⁺ regulation) and antioxidant defense (e.g., SOD activity, MDA content). This is consistent with its coastal saline habitat, where efficient Na⁺ exclusion or compartmentalization mechanisms and ROS scavenging are crucial for survival [96]. Such strategies, relying on salt glands or vacuolar sequestration, are common adaptive mechanisms in many typical halophytes [97]. In contrast, L. gmelinii demonstrated outstanding tolerance under NaHCO₃ stress, with its tolerance mechanisms more dependent on the accumulation of osmotic regulators (e.g., proline, soluble sugars) and the stability of photosynthetic pigments (e.g., chlorophyll) to cope with the combined stress of high pH and HCO₃⁻ toxicity. This strategy is typical in inland saline–alkali plants, where maintaining turgor pressure and mitigating the direct damage of high pH to photosynthetic apparatus is central to adaptation [98,99]. L. aureum exhibited moderate but stable tolerance to both stresses, lacking the extreme specialization observed in the other two species. This suggests that its tolerance may rely on the coordination of multiple physiological mechanisms rather than a single dominant pathway. Notably, the ranking of stress tolerance among species revealed by PCA is highly consistent with the saline–alkali chemical characteristics of their respective native habitats. Specifically, L. bicolor is adapted to the coastal environment with high NaCl content, L. gmelinii is specialized to the inland alkaline soils with high pH and high HCO₃⁻ content, and L. aureum occupies the transitional ecological niche of mixed saline–alkali habitats. This coordinated match among “habitat-phenotype-physiology” not only validates the ecological rationality of the results of this study but also highlights the crucial role of long-term natural selection in shaping the specific defense strategies of halophytes. The core mechanism of the adaptation strategy based on seed phenotypes revealed in this study, namely that natural selection links easily observable morphological traits to intrinsic physiological resistance, may represent an efficient strategy that has evolved repeatedly in plants adapting to adverse environments. Therefore, we speculate that this strategy has the potential to be extrapolated to a broader group of halophytes, such as Suaeda in the Chenopodiaceae family and Aeluropus in the Poaceae family.

SA and ST, as key early stress-tolerance phenotypic indicators, provide a theoretical basis for the development of rapid and non-destructive prediction tools. These easily measurable morphological traits are expected to serve as effective proxy indicators for predicting the saline–alkali tolerance potential of Limonium species and other halophytes during germination and the seedling stage. Although its predictive efficiency may vary depending on the species’ evolutionary history and life forms (such as annuals and perennials), this visible-phenotype-based approach provides a potentially universal and efficient starting point for large-scale germplasm screening. In the future, they could be applied to the preliminary screening of ornamental cultivars, or used for high-throughput pre-selection in the breeding of saline–alkali-tolerant crops and ecological grass seeds, significantly enhancing breeding efficiency. Specifically, when applying the phenotypic screening criteria established in this study to other species, it is recommended to adopt a “validate first, then apply” strategy. First, establish a local calibration curve between seed morphology and early stress resistance within the taxon, and then conduct large—scale screening. Although the results of this study support their predictive value, their universality across a broader range of taxa still requires subsequent systematic verification. The differences in early stress response strategies of the three Limonium species revealed in this study not only deepen our understanding of the adaptation mechanisms of halophytes but also have significant guiding implications for actively enhancing plant stress tolerance through biotechnological means in the future. For example, known improvement strategies such as exogenous signaling compounds (such as salicylic acid, H₂S), nanoparticles, and microbial inoculation are likely to be closely related to the regulation of the core physiological processes focused on in this study [96,97]. More importantly, the key stress-tolerance phenotypic indicators identified in this study-such as seed area (SA) and thickness (ST), as well as the early and rapidly activated ion compartmentalization and antioxidant defense, can serve as efficient biomarkers for screening and evaluating the effectiveness of the above-mentioned biotechnological strategies in the future. In future stress-resistant breeding or ecological restoration practices, these indicators can first be used to screen germplasm resources with excellent intrinsic stress-tolerance potential. Subsequently, the application of exogenous signaling substances, nano-nutrients, or specific microbial agents can be combined to maximize the colonization and growth performance of plants on saline–alkali lands. Therefore, this study has laid an important theoretical foundation for the development of precise plant stress-resistance improvement technologies guided by physiological mechanisms.

This study had certain limitations, as only seed materials from the same environmental conditions were used. To fully reflect the adaptive responses of the species in natural habitats with heterogeneous saline–alkali gradients, future research should collect seeds from wild populations distributed across habitats with different saline–alkali gradients, aiming to verify whether the interspecific differences observed under naturally heterogeneous stress conditions are consistent, thereby enhancing the ecological relevance and universality of the results. Additionally, the conclusions of this study are limited by the experimental concentration intervals; thus, we suggest that future studies adopt denser concentration gradients to precisely locate the true physiological thresholds of the species. Furthermore, long-term stress-related physiological and growth dynamics represent an important future research direction: we will extend the treatment period using lower concentrations or gradient stress, continuously monitor growth-related indicators to evaluate long-term adaptation and growth compensation effects. Meanwhile, we plan to simultaneously measure the activities of peroxidase (POD) and ascorbate peroxidase (APX) as well as their key substrates (e.g., ascorbic acid and glutathione), and supplement more comprehensive indicators related to reactive oxygen species (ROS) homeostasis, so as to systematically analyze the complete antioxidant regulatory network.

5. Conclusions

This study investigated the seed phenotypic traits of three Limonium species, as well as their seed germination, seedling growth, and physiological and biochemical indices under the stresses of sodium chloride (NaCl) and sodium bicarbonate (NaHCO₃). The results showed significant differences in the seed phenotypes of the three plants. SA and ST were identified as key seed phenotypic traits determining the tolerance to salt and alkali stresses. L. aureum exhibited the best performance in multiple phenotypic traits such as SA and ST. Under NaCl and NaHCO₃ stresses, the seed germination and seedling growth of three Limonium species were significantly affected. Specifically, during the seed germination stage, high-concentration stress led to a remarkable decline in indicators such as the germination rate. This inhibitory effect was particularly pronounced in the initial germination phase and became more intense with increasing stress levels. Although seeds retained some germination capacity after stress alleviation, excessive stress caused irreversible damage. During the seedling growth stage, the trends of plant growth indicators under different stresses diverged. Meanwhile, the ion balance of the three plants was disrupted, the content of photosynthetic pigments altered, and significant changes were also observed in the degree of membrane lipid peroxidation and the content of osmotic adjustment substances. Moreover, each species exhibited a distinct response pattern to stress.

Notably, the ranking of stress tolerance among species revealed by PCA is highly consistent with the saline–alkali chemical characteristics of their respective native habitats. Specifically, L. bicolor is adapted to the coastal environment with high NaCl content, L. gmelinii is specialized to the inland alkaline soils with high pH and high HCO₃⁻ content, and L. aureum occupies the transitional ecological niche of mixed saline–alkali habitats. This coordinated match among “habitat-phenotype-physiology” not only validates the ecological rationality of the results of this study but also highlights the crucial role of long-term natural selection in shaping the specific defense strategies of halophytes. Overall, our findings align with global research on plant saline–alkali adaptation strategies, confirming that plants exhibit fundamentally different mechanisms for adapting to neutral salts (e.g., NaCl) and alkaline salts (e.g., NaHCO₃/Na₂CO₃). This further supports the idea that plant stress resistance is not a single trait but is composed of different physiological modules driven by distinct stressors: salt tolerance is mainly driven by ion homeostasis and ROS scavenging, while alkali tolerance is closely associated with pH homeostasis, protection of photosynthetic machinery, and the synthesis of specific compatible solutes. This study also provides direct guidance for species selection in ecological restoration of saline–alkali soils. L. bicolor should be prioritized in Na⁺/Cl⁻-dominated saline soils, while L. gmelinii is more suitable for alkaline soils dominated by high pH and HCO₃⁻/CO₃²⁻ toxicity. L. aureum, with its broader adaptability, can serve as a pioneer or companion species in heterogeneous habitats with less severe or complex stresses.