## 2 Materials and Methods  
### Experimental Design  
This study selected the _Triticun aestivum_ L variety of \"Shixin 828\", extensively cultivated in the North China Plain, for outdoor pot experiments using its seeds as the culture and analyte subjects. Before planting, the seeds were screened to remove poor-quality grains, ensuring that the seeds were essentially plump. The soil used was natural farm-land soil, and the mineral components and contents of the soil samples were as follows: 66% quartz, 11% plagioclase, 6% mica, 5% calcite, 4% potassium feldspar, 3% pyroxene, 2% chlorite, 2% amphibole, and a small amount of zeolite. The basic physical and chemical properties of the soil samples were as follows: pH 8.4, organic matter 13.9 g/kg, nitrogen 830 mg/kg, fast-acting potassium 118 mg/kg and available phosphorus 11.2 mg/kg. After natural air-drying, the soil samples were cleared of gravel and plant residues, sieved through a 10 mm nylon screen, and thoroughly mixed, and then 15 kg of the original soil sample was placed into each pot of the same size. The experimental planting pots measure 35 cm in height and 32 cm in diameter, and each pot contains 15 kg of original soil samples. Following the method of controlled variable experiments, ferrous sulfate (FeSO4.7H2O) was used as the iron source to set six concentration gradient groups of iron (Figures 1 and 2), including an original soil control group. Furthermore, six parallel samples with identical soil and planting conditions were created for each gradient, resulting in a total of 36 experimental soil pots. Throughout the experiment, the planting pattern, sunlight exposure, fertilizer and water application amounts, as well as temperature and humidity within the experimental pots, were maintained under identical conditions, effectively removing external environmental interference within the controlled range. The concentration gradient configuration of the experimental soil is determined based on the effective iron content in the original soil. The third-order lower limit value of effective iron, as outlined in the Land Quality Geochemical Evaluation Specification (DZ/T 0295-2016) [34], is used as the baseline for the deficient status (Table 1), ensuring that the actual iron deficiency status in the soil can be implemented. In alkaline soil, the concentration of soluble iron in the soil solution decreases as the pH values increase. Research indicates that at higher pH levels, each unit increase in pH results in a 1000-fold reduction in active iron within the solution [35]. The extraction process in the laboratory is adequate, closely approximating the total adsorption capacity of active elements in the soil. However, the actual release amount in alkaline soil generally does not reach the extraction effect of the measured effective status. Hence, given the alkaline nature of the soil in this experiment (pH = 8.4), the available iron content in the experiment is estimated to be reduced by 5 times, resulting in levels generally lower than the critical value of deficiency lower limit. The iron concentration gradient in the experimental soil is determined based on the original third class of the effective index of soil trace elements (DZ/T 0295-2016). The average value is interpolated between the original first, second, and third classes, and then the original first class is doubled, resulting in the establishment of a new fifth-class index (Table 2). Based on the estimated available iron content in the soil, exogenously added ferrous sulfate compensation solutions were prepared at effective iron concentration levels of 0 mg/kg, 4.5 mg/kg, 7.2 mg/kg, 10 mg/kg, 15 mg/kg, and 40 mg/kg, of which 0 mg/kg serves as the control group (CK). For each concentration gradient, six samples were treated with these solutions.