Currently, soil salinization affects more than 20% of all cultivated land and approximately half of all irrigated land in the word, which will probably increase in extent and severity due to seawater intrusion and intensive irrigation practices aggravated global climate change (Hu and Schmidhalter, 2023; Hu et al., 2025) Soil salinity can harm all stages of plant growth from seed germination to reproductive stage, leading to substantial crop yield losses (Shannon, 1997; Pessarakli and Kopec, 2009). Indeed, in 2021, the global annual cost of salt-induced land degradation in irrigated areas was estimated to be US$27.3 billion related to lost crop productivity by the Food and Agriculture Organization of the United Nations (Colin et al., 2023). Therefore, it is necessary to improve the salt tolerant of modern germplasms and cultivars. This requires a comprehensive understanding of the salt tolerance mechanism in crops (Colin et al., 2023; Yang et al., 2023).

Salinity caused osmotic stress, ionic toxicity, and secondary stresses, particularly oxidative stress (Yang and Guo, 2018), together with various morphological, physiological, biochemical, and molecular changes in plants, which directly inhibit plant growth and development (Munns and Tester, 2008; Hu and Schmidhalter, 2023; Zelm et al., 2020; Hu et al., 2025). In the short-term, osmotic stress is actually caused by water deficit and inhibits the growth of new shoots and young leaves, due to the increase of salt concentration (NaCl) to a threshold level. Osmotic stress influences various aspects of plant physiology, including water absorption, seed germination, cell elongation, leaf development, lateral branching, photosynthesis rate, nutrient uptake and translocation from root to shoot, abrupt supply of carbohydrates to meristematic tissues, and ultimately exerts a negative impact on overall growth of the plant (Munns and Tester, 2008; Zelm et al., 2020). Then, at long time high salt concentration in in plant cells further generates ion toxicity. Ion toxicity of Na⁺ and Cl⁻ disrupts the uptake of essential nutrients such as Ca⁺ and K⁺, resulting in a nutritional imbalance within plants (Acosta-Motos et al., 2017).Moreover, when an excessive amount of salt ions get into the transpiration stream of plants, they cause harm to plant cells by inhibiting photosynthesis and impairing ion homeostasis (Barba-Espín et al., 2011), thus further negatively affecting plant vegetative and reproductive growth. Oxidative stress is caused by primary stresses (osmotic stress and ion toxicity) under salt stress, due to the excess production of reactive oxygen species (ROS) during metabolism. Excessive ROS synthesis can cause cell damage, such as lipid peroxidation, RNA and DNA molecules, proteins and debilitation of the metabolic processes (Mittler, 2002; Garcia de la Garma et al., 2014; Yang and Guo, 2018). Overall, salinity is responsible for different types of stresses, mainly osmotic stress, ionic stress, and oxidative stress, which together obstruct physiological and biochemical activities of the plant, and result in impairing plant growth and development.

Plants as sessile organisms cannot simply move and must cope with salt stress (Zhu, 2016). To adapt to salt stress, plants have evolved various strategies to optimize the balance between growth and stress response. The main of tolerance mechanisms is activated to ensure the maintenance of ion homeostasis in plant cells, through regulating the balance between potassium (Jadidi et al., 2022). Maintaining an optimal photosynthesis process constitutes another pivotal mechanism for achieving a high level of tolerance. Reduction in photosynthetic pigment contents and photosynthesis rate under salt stress have been used as the key biochemical and physiological indicator to screen salt-tolerant genotypes, respectively (Parihar et al., 2014). Additionally, salt treatment also induces the generation of ROS in plant cells. ROS are mainly comprised of free radicals such as O₂ ⁻ (superoxide radical), and H₂O₂ (hydrogen peroxide) (Kaushik and Aryadeep, 2014). Plants have developed a multifaceted antioxidant defense mechanism to detoxify excess ROS accumulates under salt stresses. The antioxidant defense system consists of two different types of antioxidants, namely, antioxidant enzymes (SOD, superoxide dismutase; CAT, catalase; POD, peroxidase; APX, ascorbate peroxidase, and so on) and non-enzymatic antioxidants (AsA, ascorbic acid; GSH, glutathione; CAR, carotenoids, and so on) (Gupta and Huang, 2014; Azeem et al., 2023). Increasing evidence indicates that salt stress-inducible molecular processes including numerous salt-responsive genes and transcription factors (TF) intersect with each other and form a complex network to regulate the physiological response of plants to salt stress. Ca²⁺ including two categories acts as an important pathway, which are a Ca²⁺-dependent signal transduction pathway (SOS pathway, ABA pathway, CDPK pathway) and a Ca²⁺-independent signal transduction pathway (MAPK pathway) (Hao et al., 2021; White and Broadley, 2003). All in all, there is a complex network of physiological and molecular mechanisms involved in salinity tolerance. Hence, the development of new superior salt-tolerance varieties needs a comprehensive investigation of various growth aspects and molecular (Mittler, 2002).

Wheat (Triticum aestivum L.) is one of the most important cereal crops widely planted all over the world, which is a staple food for more than one-third of the world’s people (Shiferaw et al., 2013). Considering the limited arable land area, increasing world’s population, urbanization, environmental changing, and COVID-19, wheat production cannot meet the demand of global population without a new agricultural revolution. To increase the wheat production, especially in salt affected arid and semiarid areas, creating salt-tolerant wheat cultivars is of great value. Wheat is also a moderately salt-tolerant crop that is more tolerant than rice (Oryza sativa) but less tolerant than barley (Hordeum vulgare) (Munns and Tester, 2008). It has been reported that salinity has inhibitory effects on wheat root and shoot traits, even ultimately reduces wheat production (Asgari et al., 2012). Wheat salt tolerance is a complex trait that is controlled by multiple genes and involves various biochemical and physiological mechanisms (Zhang and Shi, 2013). The performance of the morphological, physiological, biochemical parameters, and gene regulation will vary among wheat genotypes, with a genotype being superior in at least one trait and inferior in other traits under salt stress. Thus, genetic variations in salt tolerance exist and the degree of salt tolerance varies in different wheat genotypes (Naz et al., 2015). Although much has been documented about negative impacts of salt stress in wheat, its effects on morphological, physiological, biochemical, cellular and molecular mechanisms regulating plant adaptation and tolerance to salinity stress in different wheat genotypes are largely unknown (Hayat et al., 2021). In the present study, we aimed to explore the effects of salinity stress on some morphological, physiological, biochemical and cellular characteristics of wheat and also to elucidate the salt tolerance mechanism of wheat genotypes. Hence, we used a salt-tolerant wheat cultivar (QM6) and a salt-sensitive cultivar (CS) to demonstrate the different responses and adaptations to salt stress at morphological, structural, physiological, biochemical, and molecular levels. The identification of salt-tolerant mechanism in salt-tolerance wheat cultivars could represent valuable resources for genetic improvement programs to provide greater understanding of plant tolerance to salt stress, supporting agricultural production on salinized soils.

Salinity is a foremost restriction to wheat production, especially in the world’s arid and semiarid regions (Hollington, 2000). Salt stress induces a series of negative effects in morphological and structure changes with increase in salinity levels (Saddiq et al., 2021). Considering that salt tolerance is a complex phenotype determined by multiple elite alleles controlling quantitative traits, different wheat varieties have different tolerance levels under salt stress conditions (Ami et al., 2020). Many researchers examined wheat genotypes for salt tolerance have been identified through morpho-physiological traits (Majeed et al., 2019; Mahboob et al., 2018; Hasanuzzaman et al., 2022). In the present study, a winter wheat genotype (QM6) and a summer wheat (CS) were carried out a comparative study on salt tolerance at morphological, structural and molecular levels. Overall, the results showed QM6 is better suited to grow on salinized soils. Therefore, QM6 could be utilized as valuable genetic resources in wheat breeding for salinity tolerance research.

Given that the seedling stage shows the most sensitive to salinity stress throughout the plant life cycle, the salt tolerance of seedling is very important to wheat production (Ma and Weng, 2005). Salinity has adverse impacts on water and mineral uptake which leads to reduction of seedling growth (Zheng et al., 2008). The wheat seedling growth performance significantly decreased in root and shoot growth under salt stress conditions, such as shoot height, root length, and dry weight of roots and shoots (Zheng et al., 2008). Moreover, in the present study, QM6 had well-developed root system and shoot growth compared with CS after salt stress (Figures 1A, B). After NaCl treatment, the root and shoot growth of QM6 was not inhibited under low salt concentration, but was increased compared to the control (without NaCl treatment). In contrast, there was an obvious reduction in root and shoot growth of CS under different salt concentrations compared with the control (without NaCl treatment) (Figure 1). Our root growth results are in accordance with the results of our previous study (Zhang et al., 2016). However, it has been reported that winter wheat had a lower root length than spring wheat under abiotic stress, due to spring wheat with a greater supply of assimilates from leaves to growing parts (Yang et al., 2010). However, different wheat varieties have different genotypes, not all spring wheat would be expected to display salt tolerance. It is necessary to further investigate the mechanism of salinity tolerance in wheat genotypes.

Root hairs are single-cell extension from epidermal cells, which have important function in water/nutrient uptake (He et al., 2015). Development and morphogenesis of root hairs are greatly regulated by many environmental factors, such as nutrition, abiotic and biotic stresses. Recent studies have shown that plants can protect themselves by reducing the length and density of root hair and the absorption area of excessive Na⁺ when they sense stress signals (Wang et al., 2008; Liu et al., 2023). However, the differences in root hair growth between salt-tolerance wheat varieties and salt-sensitive wheat varieties under salt stress are not well understood. Here, the effect of salinity on root hair growth between QM6 (salt-tolerant) and CS (salt-sensitive) was observed via stereomicroscope and SEM. QM6 exhibited significantly more and longer root hairs in the 0.30% NaCl treatment than that in the control (Figures 2A, B), whereas there was a significant reduction in root hair density and length in CS compared with the control (Figures 2B, C). Moreover, QM6 had much more and longer root hairs under salt stress than CS (Figure 2). The results implied that the higher salt tolerance in QM6 could be attributed to its well-developed root hairs.

Salinity can also impair the normal structures and function of the organelles, such as chloroplasts. Chloroplasts are specialized plastids present in plants and algae, and their main function is photosynthesis, through which chloroplasts generate energy essential for plant growth and crop yield (Wang et al., 2024). chloroplasts also play a multitude of crucial roles beyond photosynthesis, engaging in the biosynthesis of amino acids, fatty acids, nucleotides, lipids, and vitamins, as well as the production of phytohormones, starch, and pigments (Neuhaus and Emes, 2000; Li J. Y. et al., 2022). Salinity can affect chloroplast size, number, shape, lamellar organization, starch accumulation and so on (Zahra et al., 2022). Additionally, chloroplasts are major sites for ROS production in plants subjected to salt stress (Wang et al., 2024). Thus, salt stress negatively affects the function of chloroplasts, and further impairs plant growth and development. In wheat, several changes have been associated with chloroplast structure in response to salt stress (Zuo et al., 2020). Chloroplast thylakoid membranes swelled and the accumulation of starch in the chloroplasts increased in wheat cultivars to slightly different extents under high salinity (200 mM NaCl) (Salama et al., 1994). Chloroplasts of salt-sensitive cultivars displayed an increase in volume possibly due to changes in the ionic composition of the stroma. Changes in the ionic composition of starch-degrading enzymes may also be linked with excessive starch deposition (Salama et al., 1994). Indeed, in this study, the chloroplast of QM6 possessed clearly two envelope membranes, a well-developed internal membrane system, composed of grana and long stromal thylakoids (Figure 3A). In contrast, the chloroplast shape of CS was deformed and broken (Figure 3B). Its two envelope membranes were broken and incomplete, and the grana and stoma thylakoids of chloroplast were very irregular (Figure 3B). The most important physiological process that takes place in chloroplasts is photosynthesis, which is also sensitive to salt stress (Wang et al., 2024). The initial impact of salinity on plants is osmotic stress, which leads to stomatal closure. Stomatal closure in turn impacts the process of carbon fixation during photosynthesis by limiting CO2 supply, resulting in decrease photosynthetic activity and photosynthetic rate (Amor et al., 2020). Under salt stress, the degree of photosynthetic efficiency varies differentially in diverse genetic backgrounds, which can be used as indicators to distinguish the salt tolerance of different crop genotypes (Pradhan et al., 2019). In this study, our results showed that QM6 had increased photosynthetic activity as reflected by higher Ci, Tr, Gs and Pn under salt stress conditions compared to CS. These results were consistent with previous studies, where Ci, Tr, Gs and Pn in salt-tolerant wheat varieties showed a higher level than in salt-sensitive wheat varieties (Figure 4). Taken together, chloroplast structure in our study implied that the photosynthetic activity in QM6 might be less damaged than in CS plants under salt stress. Thus, it’s necessary to further use a biparental mapping population created by crossing CS and QM6 to identify the genetic components and genes that control photosynthetic efficiency.

Salt stress induces the accumulation of ROS, which have oxidative stress-induced toxic effects on plants. ROS homeostasis is crucial for plant growth, development, and response to salt stress, but the excess production of ROS can cause toxic effects that damage all components of the cell, including proteins, lipids, and DNA, which might be corrected with plant growth and chloroplast structure in our study (Li Z. H. et al., 2022; Mittler, 2002). Detoxification signaling pathways are involved in controlling the homeostasis of cellular ROS levels under salt stress (Yang and Guo, 2018). SOD, CAT, POD and P5CS are ROS-scavenging enzyme and known to be involved in redox homeostasis of cells under various stress conditions (Zhao et al., 2022). In this study, compared to CS plants, QM6 had increased osmotic adjustment capacities and active antioxidant metabolism as reflected by higher contents of soluble sugar and proline, and higher activities of SOD and POD, respectively (Figures 5A–D). The accumulation of H₂O₂ and O₂ ⁻ in QM6 under salt stress was remarkably lower than those in CS (Figures 5E,F). Besides morpho-physiological study, previous research has established that ROS-scavenging genes and stress-responsive genes are essential in plant stress responses (Hayat et al., 2021; Çelik et al., 2019). overexpression of TaPRX-2A encoding peroxidase (PRX) improves the tolerance of wheat against salt stress (Su et al., 2020). TaSOD2 overexpression in wheat and Arabidopsis enhanced the resistance to salt and oxidative stress (Wang et al., 2016). Multiple wheat transcription factors have been identified as playing pivotal roles in enhancing plant tolerance to drought stress (Manna et al., 2021). For instance, the wheat DREB TF TaDTG6-B positively regulates TaPIF1 transcription to enhance drought tolerance in wheat (Mei et al., 2022). WRKY2 and WRKY19 positively contribute to plant tolerance to drought, salt, and cold stresses in transgenic Arabidopsis (Niu et al., 2012; Gao et al., 2018). Other stress-responsive genes, such as ERF5a and TIP2, are involved in plant response to drought and salt stress (Eini et al., 2013; Wang et al., 2011). To elucidate how QM6 promotes root and shoot growth under salt stress compared with CS, the expression patterns of some salt stress marker genes including ROS-scavenging genes (SOD, CAT, POD and P5CS) and stress-responsive genes (TaDREB1, TaDREB3, TaERF5a, TaWRKY19, TaLTP1, TaAQP7, and TaTIP2). The results showed that the ROS-scavenging genes including TaSOD1/6, TaCAT1/4/5/6, TaPOD7, and TaP5CS1/5 were upregulated in QM6 plants compared to the control under salt stress conditions. In contrast, TaSOD6, TaCAT1/5/6, TaPOD7, TaP5CS1 were dramatically downregulated in CS plants compared to the control under salt stress conditions. Furthermore, the TaP5CS5 gene expression were significantly enhanced in QM6 plants compared with CS (Figure 6A). Although the TaDREB3, TaERF5a, TaWRKY19, TaLTP1, and TaTIP2 gene expressions were all upregulated in both QM6 and CS under salt stress conditions, the change in the degree of these upregulated genes in QM6 was significantly higher than that in CS (Figure 6B). Therefore, our results indicate that QM6 had stronger adaptability to salt stress than CS probably via reducing ROS accumulation and inducing the expression of the downstream stress-related genes involved in ROS scavenging and defense mechanisms. Thus, these differentially expressed genes between CS and QM6 are likely to be candidate genes for salt tolerance, and can be developed as new diagnostic markers for marker-assisted selection in breeding programs in the future research. The application of molecular markers is productive for different genetic studies, molecular marker-assisted selection, and screening of candidate genotypes for stress tolerance. It has been used in several previous studies and reported to be very informative and highly capable to distinguish between wheat genotypes for salinity tolerance. (Shahzad et al., 2012; Elshafei et al., 2019; Hasanuzzaman et al., 2022; Al-Ashkar et al., 2020). Collectively, given that accurate phenotyping is crucial for identifying potential salt-tolerant wheat candidates, a combined approach of phenomics and genomics is vital to guarantee the successful cultivation of wheat varieties that exhibit tolerance to salinity. The tolerant and moderately tolerant genotypes have been identified as resource base population could to be utilized suitably for further improvement programme for salt tolerance in wheat.

In this study, morphological, structure, gene expression comparisons of the salt-sensitive wheat cultivar (CS) and salt-tolerant cultivar (QM6) were carried out under different salt stress levels. We have found that QM6 performed better in root and shoot growth, root hairs, chloroplast structure, and physiology in response to salinity compared with CS. Moreover, the expression level of ROS-scavenging genes and stress-responsive genes displayed more transcripts in QM6 than that in CS. Better understanding about the morphological, structural, physiological, biochemical, and molecular mechanisms of wheat varieties in response to salinity may enable further improvement in salt tolerance of wheat, and development of more salt-tolerant wheat cultivars.