Soil salinization is a major global ecological problem impacting sustainable agriculture. Cotton, known for its relatively high salt tolerance, serves as a model plant for studying salt tolerance mechanisms. However, salt-alkali stress significantly affects cotton growth. Different types of soil salinization exist, including neutral salt stress (NaCl and Na₂SO₄) characterized by osmotic stress and ion toxicity, and alkali stress (NaHCO₃ and Na₂CO₃) primarily caused by high pH disrupting ion balance and plant growth. Alkali stress may pose a greater risk than neutral salt stress. While the salt tolerance mechanisms of cotton have been studied extensively, fewer studies have compared the effects of different types of salt stress. This study aims to improve our understanding of cotton's salt tolerance mechanisms under varying salt-alkali conditions by examining the impacts on biomass, root morphology, physiological indicators (REC, MDA, proline), antioxidant enzyme activities, ionomic responses, and the expression of key salt tolerance genes (GhSOS1 and GhNHX1). Previous research indicates that salt-alkali stress inhibits cotton growth, reduces biomass, and negatively affects root development. Furthermore, ion toxicity resulting from disrupted ion homeostasis is a major consequence. Ionomics, a new approach to studying plant responses to stress, will be used to analyze the mineral nutrient and trace element ionome of cotton under different stress conditions. The interplay between mineral elements and salt stress will be crucial to understanding cotton's adaptation mechanisms.

Existing literature highlights the significant inhibitory effects of salt-alkali stress on cotton growth, reducing biomass and hindering root development. Studies have shown that salt stress (NaCl and Na₂SO₄) primarily causes osmotic stress and ion toxicity, impacting plant physiology. Alkali stress (NaHCO₃ and Na₂CO₃), characterized by high pH, interferes with plant growth by disrupting ion balance and nutrient uptake. Research indicates that the accumulation and absorption of inorganic ions such as Na+, K+, and Ca2+ contribute to osmotic adjustment and plant salinity tolerance but can also lead to ion toxicity and imbalances. Salt stress affects the uptake of both macro- and micronutrients in plants, leading to nutrient deficiency and metabolic disorders. Maintaining intracellular ionic homeostasis is a key adaptive mechanism for plants under salt stress. Two important genes involved in salt tolerance are SOS1 and NHX1. The SOS pathway maintains ionic homeostasis while NHX1 is involved in Na+ transport and compartmentalization, influencing osmotic balance. Ionomics, using techniques such as ICP-MS, provides a powerful tool for quantitatively studying the plant ionome and its response to environmental stressors.

The experiment was conducted in a greenhouse using grey desert soil and the cotton cultivar Lu-mian-yan No. 24. Three salt-alkali stress treatments were applied: NaCl (CS), Na₂SO₄ (SS), and Na₂CO₃ + NaHCO₃ (AS), with a control (CK). Soil columns were prepared, and cotton seeds were sown. After reaching the “2 leaves and 1 heart” stage, seedlings were thinned to two per column. Regular drip irrigation maintained soil moisture at 60–80% of field capacity. After 60 days, samples were collected for various analyses. Growth measurements included total biomass and root morphology (length, surface area, volume) using WinRhizo software. Physiological responses were assessed by measuring leaf REC, MDA content, and proline content. Antioxidant enzyme activities (SOD, POD, CAT) were also measured. Ionomic analysis using ICP-MS determined the concentrations of 17 elements (Na, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, B, Mo, Ni, Co, Al, Si, and Se) in leaves, stems, and roots. Gene expression analysis using RT-qPCR determined the relative expression of GhSOS1 and GhNHX1, using UBQ7 as a housekeeping gene. Statistical analysis using SPSS 21.0 included Duncan’s multiple range tests and Pearson’s correlation analyses. Principal component analysis (PCA) and hierarchical cluster analysis were performed using R software and MetaboAnalyst, respectively.

Salt-alkali stress significantly reduced cotton biomass, with the AS treatment showing the most severe reduction (58.61%). Root length, surface area, and volume were also significantly reduced under all stress treatments. MDA content and REC in leaves increased significantly under salt-alkali stress, with higher levels observed under alkali stress compared to salt stress. Antioxidant enzyme activities (SOD, POD, CAT) and proline content increased significantly under all stress treatments, suggesting an adaptive response. PCA analysis revealed distinct separation of ionome profiles under different stress conditions. Na concentration increased significantly in all organs under all stress treatments, while the concentrations of several other elements (P, K, Ca, Mg, S, Cu, etc.) decreased significantly, particularly under alkali stress. The K/Na ratio decreased significantly under all stress treatments. Correlation analysis showed complex relationships between Na and other elements. GhSOS1 expression increased under salt stress but decreased under alkali stress in leaves. GhNHX1 expression increased under salt stress in both leaves and roots.

The results indicate that cotton exhibits both tolerance and sensitivity to salt-alkali stress. The significant reduction in biomass and root development highlights the negative impact of these stresses. The increased MDA and REC levels, along with the enhanced antioxidant enzyme activities and proline content, demonstrate the plant's attempts to mitigate oxidative stress and osmotic imbalance. The observed changes in ionome profile underscore the disruption of ion homeostasis under salt-alkali stress, with alkali stress leading to a more pronounced effect on mineral nutrient absorption. The altered expression of GhSOS1 and GhNHX1 suggests a complex regulatory mechanism governing Na+ transport and homeostasis. The contrasting responses under salt and alkali stress highlight different adaptive strategies employed by cotton under these conditions. These findings contribute to a better understanding of the physiological and molecular mechanisms underlying cotton's response to salt-alkali stress.

This study demonstrates that salt-alkali stress significantly impacts cotton growth, physiological responses, and ionome. Cotton utilizes multiple mechanisms, including antioxidant enzyme systems and osmotic adjustments, to cope with stress. However, the strategies differ between neutral salt and alkali stresses. The altered expression of GhSOS1 and GhNHX1 partially explains the accumulation of Na ions under different stress conditions. Future research could focus on identifying additional genes and pathways involved in cotton's salt-alkali tolerance, developing stress-tolerant cultivars through genetic engineering, and optimizing fertilization strategies for improved cotton production in saline-alkali soils.

This study was conducted under controlled greenhouse conditions, which may not fully reflect the complexities of field conditions. The specific soil type and cultivar used may limit the generalizability of the findings. The experiment lasted for 60 days, limiting the observation of long-term effects. Further research involving field studies with diverse cultivars and soil types is needed to validate the findings and broaden their applicability.