1. Introduction
Salinity is a serious abiotic stress that already affects nearly 1.0 × 10
9 hm
2 of land worldwide, including 30% of highly irrigated land [
1]. In China, the area of salinized soil covers approximately 1.0 × 10
8 hm
2, representing almost 10% of the world’s total salinized soil area—among which, saline soil, residual saline soil, and potential saline soil account for 37%, 45%, and 18%, respectively [
2]. Excessive soil salinity can cause salt injury to plants, which manifests as damage from a variety of secondary stresses, including osmotic stress, ion toxicity, nutrient deficiency, and oxidative stress [
3]. High salt content reduces the water content of soil, causes osmotic stress, inhibits the growth and differentiation of plant organs and tissues, and may even lead to plant death [
4,
5]. Malondialdehyde and relative conductivity are important physiological indicators to measure the degree of cell membrane damage in plants under stress [
6,
7,
8]. When plants are subjected to salt stress, osmotic balance is regulated by increasing free proline content, in order to maintain the stability of enzyme structures and the integrity of the membrane system [
9]. Under salt stress, salt-tolerant plants can selectively absorb K
+ and transport it to their above-ground organs, simultaneously reducing the transport of Na
+ to their above-ground organs, thus maintaining a higher K
+/Na
+ ratio in order to reduce the damage of salt stress on the above-ground tissues [
10,
11,
12].
The saline–alkali land in China is mainly distributed between Inner Mongolia, Gansu, Qinghai, Tibet, and Xinjiang. The Qinghai–Tibet Plateau (QTP) is a large area of saline soil distribution in China [
13]. The introduction and the planting of vegetation restoration are dependent on the biological improvement and utilization of saline soil [
14]. Siberian wildrye (
Elymus sibiricus L.) is the typical plant of the
Elymus genus in the Triticeae of the Poaceae [
15]. It is an allotetraploid perennial herb (genome constitute is StStHH, 2n = 4x = 28). It is one of the endemic species of the QTP. It can form dominant species or constructive species in meadow and grassland plant communities.
E. sibiricus has been recognized for its remarkable attributes, including elevated crude protein content; desirable palatability; vigorous tillering capacity; superior yield potential; straightforward cultivation techniques; and remarkable resilience towards cold and drought conditions [
16,
17,
18,
19]. Moreover, owing to its extensive root system and adaptability, certain
E. sibiricus germplasms have shown promising prospects for cultivation in saline–alkali environments [
20]. Therefore, understanding the physiological resistance mechanisms of
E. sibiricus with regards to salinity tolerance and using it for saline land management and improvement is a cost-effective strategy for saline land management in China.
In this study, the salt response of 50 E. sibiricus wild accessions was evaluated by measuring their relative water content (RWC), relative electrical conductivity (REC), malondialdehyde content (MDA), proline content (Pro), and Na+/K+ ratio after two weeks of salt stress treatment on seedlings. The aims of this study were to improve the salt tolerance evaluation framework of E. sibiricus, to assess the salt tolerance of these E. sibiricus accessions, and to identify robust salt stress tolerant germplasm. This study can provide the genetic resources available for breeding new salt-tolerant E. sibiricus varieties, and offer insights that can inform broader plant breeding and stress tolerance studies of endemic species to the QTP.
4. Discussion
Currently, soil salinity is one of the environmental factors affecting plant growth, causing secondary stresses like ionic, osmotic, and oxidative stress, which inhibit the growth and differentiation of plant tissues and organs, causing plant leaves to wilt and curl, and plants to wilt and dwarf [
27,
28,
29,
30,
31]. Water molecules are abundant in plant leaves, and the amount of water content in leaves is a key factor in determining plant survival, as well as being an important indicator of overall plant water content, which is extremely important in assessing the physiological status of plants under salt stress. Leaf water content was negatively correlated with the salinity of the growing environment—the higher the salinity, the more significant the effect [
32]. In an analysis of the salt tolerance of alfalfa, it was found that the variation of leaf water content varied among different varieties of alfalfa, and its salt tolerance was positively correlated with leaf water content [
33]. In this study, the relative water content of
E. sibiricus leaves varied widely (68.61% to 95.24%) under salt stress, indicating that different genotypic materials showed large differences in relative water content under salt stress, which can provide preparation for the screening of different salt-tolerant germplasm materials. It was found that the
Triticosecale rimpaui germplasm accessions with higher salt tolerance could maintain higher relative water content under salt stress, indicating that it is important for plants to maintain and utilize internal water under salt stress [
34].
The cytoplasm of plant leaf cells is tightly bound to the cell wall, forming a membrane known as the leaf cell membrane. When plants are in a high salinity environment, this membrane increases its permeability due to varying degrees of damage, leading to extravasation of intracellular electrolytes, resulting in an increase in the conductivity of the external fluid and a consequent increase in the relative conductivity. The accumulation of Malondialdehyde (MDA) indicates a level of damage to membranes and cells. MDA is a measure of membrane damage and is the end product of lipid peroxidation, and its values are useful in assessing the extent of plant damage due to stress [
6,
7,
8]. In the salt stress experiment of
Chenopodium quinoa Willd [
35], the MDA content of leaves increased significantly, aggravating the degree of membrane ester peroxidation and causing cell membrane damage. The changes in MDA levels reflect the peroxidation of plant cytoplasmic membranes under stress. It is generally believed that the higher the intracellular MDA content, the greater the damage to the membrane system. In the present study, different
E. sibiricus materials showed different levels of malondialdehyde response, with a minimum of 3.75 nmol g
−1 and a maximum of 40.00 nmol g
−1, which provided some basis for the salt tolerance evaluation work. To maintain cell expansion pressure and alleviate osmotic stress, plants also actively accumulate organic substances for osmoregulation. Proline is the most important osmoprotectant for plants to regulate osmotic potential in response to stress [
36]. It has been shown that both
E. sibiricus and
Elymus nutans have adapted to saline environments by accumulating proline [
37]. In the present study, the proline accumulation of
E. sibiricus in a high salinity environment ranged from 6.28 μg g
−1 to 49.67 μg g
−1, and the materials with high proline content grew well, indicating that
E. sibiricus can regulate cellular osmotic potential and mitigate osmotic damage by accumulating large amounts of proline. Accumulating more proline may be one of the important strategies for
E. sibiricus to respond to salt stress.
Excessive sodium ions under salt stress can lead to ion imbalance, which can cause ion toxicity because most salt-intolerant species do not have the ability to translocate Na
+, and Na
+ is more likely to accumulate in plants compared to CI
−. While K
+ is an important ion involved in plant photosynthesis, osmoregulation, and other processes in plants, too much Na
+ inhibits K
+ uptake, which in turn affects plant growth. Therefore, plants maintain a low Na
+/K
+ ratio as a reflection of their high salt tolerance [
38]. This phenomenon has been observed in rice [
39], oats [
40], and alfalfa [
41]. The Na
+/K
+ ratio of the test materials varied from 0.22 to 1, with an average value of 0.61. In general, the salt tolerance of different germplasm differed significantly. The highest Na
+/K
+ ratio was 1, which was lower than that of oats [
42], indicating that
E. sibiricus species have strong salt tolerance.
Salt tolerance in plants is a quantitative genetic trait, controlled by multiple genes that reflect the genetics, physiology, and biochemistry of the plant’s own responses. The strength of salt tolerance cannot be accurately evaluated by only one indicator, so a comprehensive evaluation of indicators should be conducted when studying the salt tolerance of plants [
36]. The fuzzy mathematical affiliation function method is widely used to evaluate the salt tolerance of many plants because it can eliminate the one-sidedness brought by a single index [
40,
41,
42]. Due to the correlation between different indicators, the salt tolerance information overlaps, and it is necessary to determine the positivity and negativity of the affiliation function, although salt tolerance is a comprehensive trait, and most of the time, it is not a simple linear relationship [
43,
44]. Therefore, the present study was conducted by principal component analysis. Principal component analysis was used to convert each individual index of
E. sibiricus into three independent composite indices that reflect most of the information of the indices. These were combined with the weights to calculate the comprehensive evaluation value of drought tolerance (D values) in order to identify the salt tolerance of
E. sibiricus, and then combined with the results of conventional affiliation function analysis based on the measured indices to evaluate the salt tolerance of
E. sibiricus. The accuracy of the study results was improved by combining the results of the conventional affiliation function analysis with multiple evaluation methods.
5. Conclusions
In this study, salt tolerance evaluation was conducted on diverse geographical sources of collected E. sibiricus germplasm. Multiple assessment methods were employed, revealing that there was no significant correlation between geographic origin and salt tolerance. This lack of correlation could be attributed to the sampling strategy being random in nature and the limited diversity of accessions. Through comprehensive evaluation, salt-tolerant germplasm was successfully identified, providing a valuable reference for the assessment of highly resistant germplasm in E. sibiricus and its related species. Additionally, in future salt tolerance evaluations, it is recommended to utilize various assessment methods, such as membership functions and PCA, focusing on indicators such as REC, Pro, Na+/K+, MDA, and others. Moreover, this evaluation provides useful resources for the development of grazing pastures and artificial grasslands in challenging environmental conditions.