Mechanism of Iron Transport in the Triticum aestivum L.–Soil System: Perception from a Pot Experiment

Abstract

Iron is one of the necessary trace elements for plant growth and the human body. The ‘hidden hunger’ phenomenon in the human body caused by an imbalance of iron in soil is increasingly prominent. Addressing this issue and optimizing soil through regulatory measures to improve the absorption and utilization of iron by crops has become an urgent priority in agricultural development. This study carries out pot experiments to observe the growth process of Triticum aestivum L. under various soil iron environments. Combined with previous research results, the transport mechanism of iron in the soil—Triticum aestivum L. system was systematically explored. The results indicate that during the jointing and maturity stages of Triticum aestivum L., iron was preferentially enriched in the underground parts; at the maturity stage, the iron content in various organs of Triticum aestivum L. shows a trend of increase followed by a decrease with the soil iron content varying in the following sequence: deficient, moderately deficient, medium, moderately adequate, and adequate. The iron-deficient stress environment causes an increase in the effectiveness of rhizosphere iron, resulting in a higher level of iron in the plant stems, leaves, and seeds. Conversely, when the soil iron content is medium or adequate, the effectiveness of rhizosphere iron decreases, leading to a reduction in the iron content in each part of the plant. A concentration gradient of 7.2 mg/kg in the experimental setup is found to be the most favorable to the enrichment of iron in the shoots of Triticum aestivum L. plants. The findings of this experiment provide guidance for the fertilization strategy to mitigate iron deficiency symptoms in plants under similar acidic-alkaline conditions of soil, as well as a systematic mechanism reference and basis for studying the soil-plant-human health relationship.

1. Introduction

One of humanity’s greatest challenges is how to sustainably feed a large population, especially in China [1,2]. Triticum aestivum L. is one of the most important grain crops in the world [3], and increasing its yield is an effective way to solve the problem of food shortage caused by rapid population growth [4,5]. As crop yields continue to improve, the usage of traditional organic fertilizers to increase the availability of major nutrients (N, P, and K) has been increasing. However, effective supplementation of trace nutrients necessary for plant growth (particularly for iron) is neglected in large production systems [6,7,8]. The utilization of these conventional fertilizers, which usually lack iron, can lead to iron deficiency in the soil-plant system. Such a deficiency not only decreases agricultural yields but also impacts human health through “hidden hunger”, an insidious form of micronutrient (Fe) deficiency that can hinder growth, weaken immune function, and lead to anemia [9,10]. Iron deficiency in soil-plant systems is a widespread challenge in agricultural production worldwide and a scientific issue that needs to be further addressed.

Iron is a metal element that is widely distributed in nature, with a high abundance in the Earth’s crust. The total iron content in soil typically ranges from 1% to 10%. However, it is not readily available because of its low solubility [11]. According to statistics, potential iron deficiency affects approximately 40% of the world’s arable land [12]. Iron is an essential trace mineral element for the growth and development of plants. It serves as an integral component of vital enzymes, including cytochrome, cytochrome oxidase, and catalase [13]. When the soil is deficient in iron, it significantly impacts the growth and development of crops [14], leading to reduced yield and quality [15]. Two methods, leaf spraying iron fertilizer and soil micro-fertilizer, are commonly employed to resolve the symptoms of iron deficiency in Triticum aestivum L. in soils. It has been shown that the micro-fertilizer of iron carrier chelates is not affected by the pH value and Ca++ ion concentration of soil medium, which is of great significance for calcareous and alkaline soils [2,16]. However, excessive application of soil micro-fertilizer can result in resource wastage and environmental quality issues such as heavy metal pollution in surface soil and contamination of shallow groundwater, posing health risks [17]. For example, excessive iron contamination is widespread (>0.3 mg/L) [18]. Over 50 mg/L iron will cause toxicity to microorganisms and significantly inhibit microbial activity [19]. The threshold of soil micro-fertilizer usage to ensure the improvement of soil quality without wasting resources remains unknown.

The iron ion has an 18-electron shell, and its high electrostatic field makes it readily form complex ions [20]. To date, the physiological mechanism of iron uptake and utilization by plants has been extensively documented in the literature [21,22,23,24,25]. Romheld was the first to propose the Mechanism I and Mechanism II systems of iron uptake for higher plants under iron deficiency [26]. The typical Triticum aestivum L. vegetation studied in this research belongs to mechanism II plants, which are herbaceous monocotyledonous grasses capable of adapting to high-pH soil. In an iron-deficient environment, these plants can induce the production of high-iron transporters (MAs) in the Triticum aestivum L. rhizosphere and secrete them to activate insoluble iron in the rhizosphere, forming a stable octahedral Fe³⁺ chelate, i.e., Fe³⁺-MAs, which is then specifically absorbed by the rhizosphere to adapt to iron deficiency. After the plant root system absorbs iron from the environment, the plant senses its iron nutritional status and regulates the distribution of iron through its own signaling and regulatory system [23]. Previous research has indicated that iron typically exists in plants in the form of chelates. The primary iron-containing chelates in plants include citric acid [27,28], nicotinamide [29] and mugineic acids [30]. The transporter citric acid facilitates the transportation of iron from roots to overground parts. Iron obtained from plant roots can be transported to overground parts in the form of citric acid-iron trivalent chelate through xylem for long distances [31], and this process is achieved through the xylem [32]. Iron is transported from old leaves to new leaves by iron transporters in the phloem. Various types of iron transporters in the phloem, such as iron transporter proteins and nicotinamides, have been identified in multiple studies, specifically binding with ferric ions [33].

In summary, based on the research on the biological mechanisms of soil-plant interaction promoting nutrient efficiency and yield improvement, we observed the growth process of Triticum aestivum L. under different iron gradients through pot experimental systems, and further systematically explored the transport mechanism of iron in the soil–Triticum aestivum L. system. A measure of 7.2 mg/kg of iron is found to be the most favorable to the enrichment of iron in the shoots of Triticum aestivum L. plants. This study not only provides an innovative perspective and approach to reveal the promotion of nutrient efficiency plant-soil systems but also provides a scientific theoretical basis for the study of the soil-plant-human health relationship. In addition, this study provides theoretical and technical support for the development of green intelligent trace element fertilizer to promote the development of green agriculture.

2. Materials and Methods

2.1. Experimental Design

This study selected the Triticum 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 farmland 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 (Figure 1 and Figure 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. Grade index of available iron in soil (based on DZ/T 0295-2016 appendix D1).

Indices	Class A (Adequate)	Class B (Moderately Adequate)	Class C (Medium)	Class D (Moderately Deficient)	Class E (Deficient)
Available iron (mg/kg)	>20	>10~20	>4.5~10	>2.5~4.5	≤2.5

), 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. Grade index of available iron concentration gradient in the experimental soil.

Indices	Class A (Adequate)	Class B (Moderately Adequate)	Class C (Medium)	Class D (Moderately Deficient)	Class E (Deficient)
Available iron (mg/kg)	>40	>15~40	>10~15	>7.2~10	≤4.5

). 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. The ferrous sulfate compensation solution contains Fe²⁺ as reduced iron ions, which are easily oxidized to Fe³⁺ in the soil solution, and Fe³⁺ tends to form ferric hydroxide precipitates, limiting the availability of iron ions for plant uptake. In this experiment, the compensation solution is chelated with citric acid and the pH is adjusted to neutral with sodium carbonate before being added to the soil. The chelation of citric acid with iron forms a soluble iron complex, facilitating the mobility of iron and allowing iron ions to diffuse to the roots for plant uptake. The soil was uniformly mixed and allowed to stand until it was dry and free from muddy clumps before evenly planting Triticum aestivum L. in loosened, leveled soil.

The sample data collection for this experiment is primarily divided into two stages: the jointing stage and the mature stage of Triticum aestivum L. growth. At the jointing stage, five Triticum aestivum L. seedlings and their root soil were randomly chosen from each experimental pot and sent to the laboratory for the analyses of the total and effective iron content in the root soils and the iron content in the Triticum aestivum L. roots, stems and leaves. At the mature stage, all the Triticum aestivum L. seedlings were initially collected from the individual experimental pots. As the characteristics of Triticum aestivum L. growth changed, the sampling parts were adjusted to include the root soil, Triticum aestivum L. roots, stems, and ears (seeds with hulls). In the process of the experiment, the elevated outdoor temperatures during the grain filling stage of Triticum aestivum L. partially hindered the photosynthesis of Triticum aestivum L. leaves and affected the synthesis and accumulation of organic matter. Meanwhile, high nighttime temperatures accelerate Triticum aestivum L. respiration, resulting in increased nutrient consumption, insufficient grain filling, and reduced yield. To ensure the grain weight of the Triticum aestivum L. ear sample, the test sample at the final maturity stage consists of seeds with hulls. Once the samples are collected, the plant samples are promptly separated into different parts (roots, stems, leaves, and seeds), air-dried, and then sent to the laboratory for testing.

2.2. Solution Preparation

First of all, the compensation concentration values that should be applied at different gradient levels were calculated using the following formula:

$${\mathrm{F}\mathrm{e}\mathrm{C}}_{\mathrm{x}}={\mathrm{C}}_{\mathrm{F}\mathrm{e}\mathrm{x}}-{\mathrm{C}}_{\mathrm{F}\mathrm{e}0}$$

(1)

where ${\mathrm{F}\mathrm{e}\mathrm{C}}_{\mathrm{x}}$ represents the compensation concentration value that should be applied to the x-th concentration gradient of Fe, ${\mathrm{C}}_{\mathrm{F}\mathrm{e}\mathrm{x}}$ represents the soil standard concentration value corresponding to the x-th concentration gradient of Fe in Table 2, ${\mathrm{C}}_{\mathrm{F}\mathrm{e}0}$ represents the available iron content in the soil (i.e., the presumed available iron content in soil for the control group), and $\mathrm{x}$($\mathrm{x}=1,2,\dots ,5$) represents the corresponding gradient level of the element.

In addition, the concentration of solution to be applied to make up for the compensation concentration (${\mathrm{F}\mathrm{e}\mathrm{C}}_{\mathrm{s}\mathrm{x}}$) was calculated using the following formula:

$${\mathrm{F}\mathrm{e}}_{\mathrm{M}}\times {\mathrm{F}\mathrm{e}\mathrm{C}}_{\mathrm{s}\mathrm{x}}={\mathrm{S}}_{\mathrm{M}}\times {\mathrm{F}\mathrm{e}\mathrm{C}}_{\mathrm{x}}$$

(2)

where ${\mathrm{F}\mathrm{e}}_{\mathrm{M}}$ represents the amount of solution applied per pot for each application, calculated as 1.5 kg, ${\mathrm{F}\mathrm{e}\mathrm{C}}_{\mathrm{s}\mathrm{x}}$ represents the application concentration required to make up the compensation for the x-th concentration gradient of Fe, and ${\mathrm{S}}_{\mathrm{M}}$ represents the dry weight of the experimental soil, calculated as 15 kg dry weight. From this, the concentration of each micronutrient application level was formulated (

Table 3. Calculation of concentration of applied solution (based on 1. 5 kg of liquid per application, unit mg/kg).

Indicator	Measured Available Content in Soil	Presumed Available Content in Soil	Solution Preparation
Available iron (mg/kg)	11.9	2.38	Target concentration	40	15	10	7.2	4.5
			Compensation concentration	37.62	12.62	7.62	4.82	2.12
			Application concentration	376.2	126.2	76.2	48.2	21.2

).

Based on the above-calculated concentration of each class of application solution, the amount of ferrous sulfate needed for each class was calculated by applying 1.5 kg of solution per pot, and totaling 9 kg of application solution for each class of 6 pots, taking into account of factors such as spraying losses (

Table 4. Weights of materials used for each 1 kg of solution preparation (mg).

Element	Raw Materials			Class A	Class B	Class C	Class D	Class E
	Compound	Mol. Formula	At.w./ Cont. %	Conc./Weight	Conc./Weight	Conc./Weight	Conc./Weight	Conc./Weight
Available iron	Fe(II) sulfate	FeSO4·7H2O	55.85	376.2	126.2	76.2	48.2	21.2
	M.W.	278.05	20.09	1872.57	628.17	379.29	239.92	105.53

Cont.: content, Conc.: concentration, M.W.: molecular weight.

). In order to fully facilitate the absorption of plant roots, citric acid must be used to prepare complex concentrated solution, which will be diluted when applying. The specific operation is as follows: weigh the required weight of each class of ferrous sulfate raw material and put it in a 500 mL beaker (weighed clean and dry beaker in advance, accurate to 0.001 g), add an appropriate amount of water to dissolve, then add citric acid for complexation, and then gradually add sodium carbonate to adjust the pH value to 5–7 after complete dissolution; finally, place the beaker on a balance and make up for the weight of concentrated solution with water.

2.3. Sample Analysis

The sample analysis was conducted by Hebei Geological Testing and Analysis Center. The total iron content in soil samples was determined using an X-ray fluorescence spectrometer (ZSX Primus II, Rigaku Corporation, Tokyo, Japan). The available iron in soil was determined using the DTPA extraction method; extracts were analyzed using an inductively coupled plasma mass spectrometer (ICP-MS, model Agilent 725, Agilent Technologies, Santa Clara, CA, USA). The pH was determined using the glass electrode method, with measurements performed using the PHS-3C pH meter produced by Shanghai Kangyi Instrument Co., Ltd., Shanghai, China. The iron content in plants was determined in accordance with the method requirements outlined in the National Food Safety Standard—Determination of Multiple Elements in Foods (GB5009.268-2016) [36]. The analysis was performed using an inductively coupled plasma mass spectrometer (ICP-MS, model Agilent 7900, Agilent Technologies, Santa Clara, CA, USA). The complete elemental analysis utilizes first-class national reference materials for quality control, ensuring that the analytical data reportable rate, accuracy, and precision qualification rate all achieve 100%.

2.4. Evaluation Indicators

2.4.1. Enrichment Coefficient

The enrichment coefficient is the ratio of the concentration of elements in a specific part of a plant to the concentration of the same elements in the soil in which the plant grows. It is an essential index for describing the accumulation trend of chemical substances in organisms and, to some extent, reflects the challenges associated with elements migrating from sediments or soil to plants [37,38].

The coefficient is formulated as follows:

enrichment coefficient = C_plant/C_soil

(3)

where C_plant represents the concentration of elements in roots, stems or leaves of plants, and C_soil represents the concentration of the corresponding elements in soil, measured in mg/kg.

2.4.2. Translocation Factor

The translocation factor is defined as the ratio of the concentration of elements in the overground parts of plants to the corresponding concentration of elements in the underground parts of plants. It serves as a crucial index for describing the translocation of chemical substances within organisms [39], and to a certain extent, reflects the mobility of elements from the plant root to the overground part.

The factor is formulated as:

translocation factor = C_overground/C_underground

(4)

where C_overground is the sum of concentrations of elements in roots, stems or leaves of plants, and C_underground is the concentration of corresponding elements in soil, measured in mg/kg.

2.5. Data Processing

The experimental data were statistically analyzed and organized using Excel 2010. The significance of the differences between means was tested using Tukey’s Honest Significant Difference (HSD) method, and statistical significance letters were assigned. Correlation analysis and graphing were completed using SPSS 20.0 software.

3. Results

3.1. Characteristics of Element Contents in Soil and Different Parts of Triticum aestivum L.

In this Triticum aestivum L. pot experiment, the roots, stems and leaves of Triticum aestivum L. at the jointing stage as well as the roots, stems and grains at the mature stage were collected for measurements of the total and available iron contents in the soil and the iron content in the Triticum aestivum L. roots, stems, leaves, and grains during various growth periods. The statistical results are listed in

Table 5. Statistical characteristics of iron contents in soil and different parts of Triticum aestivum L. in the pot experiment.

Growth Stage	Analyte	Minimum	Maximum	Mean	Standard Deviation
The jointing stage (n = 36)	Total iron in soil	3.82	3.93	3.87	0.03
	Available iron in soil	9.70	30.40	15.13	5.21
	Root	592	3437	1262	551
	Stem	86	872	179	131
	Leaf	112	401	175	61
The maturity stage (n = 36)	Total iron in soil	3.64	3.75	3.70	0.03
	Available iron in soil	10.20	18.60	13.13	2.26
	Root	801	3848	1661	693
	Stem	187	1017	484	215
	Seed	93	216	141	29

Note: The total iron content in the table is expressed in %, while the other units are in mg/kg.

. The results show that the average iron accumulation values in the roots, stems, and leaves of Triticum aestivum L. at the jointing stage are 1262, 179, and 175 mg/kg, respectively, suggesting that iron accumulation in the roots is significantly higher compared to the stems and leaves; the average iron accumulation in the roots, stems, and seeds of Triticum aestivum L. at the maturity stage is 1661, 484, and 141 mg/kg, respectively, with the order of accumulation being roots > stems > seeds. Iron accumulation in the roots is highest at different growth stages, and the iron content in roots and stems at the maturity stage is higher than that at the jointing stage, while iron accumulation in grains is the lowest. The total and effective iron contents in soil are higher at the jointing stage than those at the maturity stage.

3.2. Correlation Analysis between Total Element Contents in Soil and Specific Contents in Various Parts of Triticum aestivum L.

3.2.1. The Jointing Stage

As shown in

Table 6. Correlation analysis between the total and effective iron contents in soil and the iron contents in different parts of Triticum aestivum L. at the jointing stage (Pearson correlation coefficient, n = 36).

Iron Content	Total Iron	Available Iron	Root	Stem
Total iron	1	0.691 **	−0.110	−0.271
Available iron	0.691 **	1	−0.058	−0.252
Root	−0.110	−0.058	1	0.039
Stem	−0.271	−0.252	0.039	1
Leaf	0.504 **	0.626 **	0.007	−0.086

** A significant correlation at the 0.01 level (two-tailed).

, the correlation analysis between the total and effective Fe contents in soil and the Fe contents in roots, stems and leaves of Triticum aestivum L. at the jointing stage reveals an extremely significant correlation (p < 0.01), with a Pearson correlation coefficient of 0.691. The correlation coefficient values are high between the iron content in Triticum aestivum L. leaves and the total iron and effective iron content in soil, with correlation coefficients of 0.504 and 0.626, respectively, indicating a significant correlation. However, the correlation between the iron content in roots and stems and the iron content in soil is poor. The jointing stage is the vigorous growth period of Triticum aestivum L., and the reproductive growth gradually intensifies. During the jointing stage of Triticum aestivum L., the transport direction of Fe element is mainly from soil to the leaf tip of the growing plant, and the Fe uptake in leaves mainly comes from available Fe in the soil.

3.2.2. The Maturity Stage

It can be seen from

Table 7. Correlation analysis between the total and available iron content in soil and the iron content in different parts of Triticum aestivum L. at the maturity stage (Pearson correlation coefficient, n = 36).

Iron Content	Total Iron	Available Iron	Root	Stem	Seed
Total iron	1	−0.079	−0.143	−0.033	−0.102
Available iron	−0.079	1	0.141	0.096	−0.351 *
Root	−0.143	0.141	1	0.638 **	−0.153
Stem	−0.033	0.096	0.638 **	1	−0.303
Seed	−0.102	−0.351 *	−0.153	−0.303	1

* A significant correlation at the 0.05 level (two-tailed). ** A significant correlation at the 0.01 level (two-tailed).

that during the maturity stage of Triticum aestivum L. growth, the correlation coefficient between available iron in soil and seeds was 0.351, reaching the significant correlation level in terms of the Pearson correlation coefficient (p < 0.05). This suggests that the iron element in seeds primarily originates from the soil. The concentration of effective iron in soil directly influences the iron content in seeds. At the same time, the correlation coefficient between Triticum aestivum L. roots and stems is 0.638, indicating a significant correlation (p < 0.01).

3.3. Variation in Element Contents in Roots, Stems, Leaves and Grains of Triticum aestivum L.

The variations in iron content in different organs of Triticum aestivum L. at different growth stages were obtained from six concentration gradients in the Triticum aestivum L. pot experiment, as illustrated in

Table 8. Distribution characteristics of iron content in organs of Triticum aestivum L. during jointing and maturity stages.

Concentration Gradient (mg/kg)		The Jointing Stage			The Maturity Stage
		Roots	Stems	Leaves	Roots	Stems	Seeds
CK (n = 6)	Minimum	918	120	118	816	193	113
	Maximum	3437	306	164	1398	735	180
	Mean	1649	227	142	1098	467	155
	Standard Deviation	945	72	17	261	176	23
	Sig.	a	ab	b	c	ab	a
4.5 (n = 6)	Minimum	763	112	139	863	187	114
	Maximum	1932	212	240	1778	724	155
	Mean	1152	172	177	1364	417	133
	Standard Deviation	426	38	35	383	176	16
	Sig.	ab	ab	b	c	ab	a
7.2 (n = 6)	Minimum	676	135	113	926	501	112
	Maximum	1623	872	222	2926	830	188
	Mean	1145	288	162	2206	612	151
	Standard Deviation	388	288	40	767	166	28
	Sig.	ab	a	b	a	a	a
10 (n = 6)	Minimum	592	88	112	871	225	97
	Maximum	1177	189	201	3848	1017	216
	Mean	975	143	156	2142	553	149
	Standard Deviation	206	45	35	1067	342	45
	Sig.	b	ab	b	ab	ab	a
15 (n = 6)	Minimum	802	97	114	801	267	105
	Maximum	1780	196	190	1764	620	181
	Mean	1167	127	143	1444	353	134
	Standard Deviation	330	36	30	370	134	28
	Sig.	ab	b	b	bc	b	a
40 (n = 6)	Minimum	751	86	192	1257	281	93
	Maximum	2429	180	401	2209	848	150
	Mean	1481	116	271	1709	505	127
	Standard Deviation	609	33	83	320	217	24
	Sig.	ab	b	a	abc	ab	a

Note: Statistical significance of means was tested using Tukey’s Honest Significant Difference (HSD) test. In the table, “Sig.” denotes the significance of the Tukey statistical test (p < 0.05). The letter assignment for statistical significance was as follows: the means in each column were arranged from largest to smallest. The highest mean was designated as ‘a’, and this mean was compared with each subsequent mean. Means not significantly different were also marked as ‘a’. Significantly different means were marked as ‘b’. The mean marked ‘b’ was then used as the standard for comparison with larger means, marking means not significantly different as ‘b’ until a significant difference was found. This process continued with subsequent letters (‘c’, etc.) until all means in the column were assigned.

and Figure 3. At the jointing stage, the iron content in the leaves did not significantly differ under various soil conditions, ranging from deficient to adequate available iron backgrounds, and it was only significantly higher than the normal value in the adequate background (Figure 3c). The iron content in the stem generally decreased with the increase in the iron ion concentration in the soil (Figure 3b). Meanwhile, the iron content in the roots displayed a pattern of initially decreasing and then increasing, with the lowest mean value occurring when the iron ion concentration gradient in the soil was at a medium level, i.e., 10 mg/kg level (Figure 3a). At the maturity stage, the iron content in roots, stems, and seeds initially increased and then decreased with the rise in iron concentration in the soil (as shown in Figure 3d–f). At a moderately deficient iron ion concentration gradient in the soil (at the 7.2 mg/kg level), the iron content in roots, stems, and seeds reached its peak, with the average iron content in roots recorded at 2206 mg/kg, in stems at 612 mg/kg, and in seeds at 151 mg/kg.

The iron content in the roots of Triticum aestivum L. at the jointing and mature stages is significantly higher compared to the stems, leaves, and seeds under the treatment of six different iron concentration gradients in the experimental soil. This suggests that the majority of the iron absorbed by Triticum aestivum L. roots is accumulated in the roots, with only a small portion being transported to the overground parts. Throughout the Triticum aestivum L. growth period, the continuous supply of rhizosphere iron resulted in higher iron content in the roots at the maturity stage than at the jointing stage, and except the control group, the variation trend of the rhizosphere iron is the most significant under iron deficiency stress conditions.

3.4. Enrichment Coefficients of Iron in Different Parts of Enrichment Coefficients of Iron in Different Parts of Triticum aestivum L.

The enrichment coefficients of iron in different parts of the Triticum aestivum L. plants in this experiment are presented in

Table 9. Enrichment coefficient and translocation factor of iron in Triticum aestivum L. plants at the jointing stage.

Concentration Gradient	Enrichment Coefficient of Root	Enrichment Coefficient of Stem	Enrichment Coefficient of Leaf	Underground to Aboveground Translocation Factor
CK	0.043	0.006	0.004	0.286
4.5 mg/kg	0.030	0.004	0.005	0.321
7.2 mg/kg	0.030	0.007	0.004	0.393
10 mg/kg	0.025	0.004	0.004	0.330
15 mg/kg	0.030	0.003	0.004	0.248
40 mg/kg	0.038	0.003	0.007	0.317
Mean	0.033	0.005	0.005	0.316

and

Table 10. Enrichment coefficient and translocation factor of iron in Triticum aestivum L. plants at the maturity stage.

Concentration Gradient	Enrichment Coefficient of Root	Enrichment Coefficient of Stem/Leaf	Enrichment Coefficient of Seed	Underground to Aboveground Translocation Factor
CK	0.030	0.013	0.004	0.570
4.5 mg/kg	0.037	0.011	0.004	0.415
7.2 mg/kg	0.060	0.017	0.004	0.389
10 mg/kg	0.058	0.015	0.004	0.352
15 mg/kg	0.039	0.010	0.004	0.356
40 mg/kg	0.046	0.014	0.003	0.381
Mean	0.045	0.013	0.004	0.411

. It can be seen from the table that the enrichment coefficients of different parts of Triticum aestivum L. are all less than 1, showing an enrichment pattern of roots > stems/leaves > seeds. The enrichment coefficient of iron in Triticum aestivum L. roots is considerably higher than that in overground parts, with the average enrichment coefficient of roots being 0.033 at the jointing stage and 0.045 at the maturity stage, respectively. The enrichment coefficients of stems, leaves, and seeds at the jointing stage are similar, averaging 0.005. At maturity, the enrichment coefficient of the stem and leaf increased by one order of magnitude, reaching an average value of 0.013. The enrichment coefficient of seeds is low, differing by one order of magnitude from that of roots and stems. Conversely, the enrichment coefficient remains largely consistent across different concentration gradients of soil iron. The enrichment coefficient characteristics mentioned above indicate that adjusting the background iron content in the soil has a minimal impact on Triticum aestivum L. seeds.

4. Discussion

4.1. Mechanism of Iron Transport in Soil and Triticum aestivum L. Plants

The experimental findings indicate that both the total and effective iron contents in the soil decrease as Triticum aestivum L. grows, whereas the iron content in various parts of the plant continues to accumulate. The correlation analysis indicates that the available iron in soil predominantly influences the aboveground portions of the plant, such as leaves and grains, signifying the migration of iron between the soil and the Triticum aestivum L. plants.

In this pot experiment, citric acid was used as a chelating agent ligand. Based on previous data [40], it is presumed that the transport mechanism of iron between the soil and the Triticum aestivum L. plants involves the formation of a cyclic chelate between citric acid and iron ions through carboxyl (COOH) and hydroxyl (OH) coordination groups and its own carbon chain. The strong coordination ability of iron ions enables them to form coordination with the carboxyl and hydroxyl groups in the citric acid ligand. Upon adhering to the root surface, iron ions undergo reduction and degradation on the root surface as a result of the release of H+ and e- by the root system. The iron element is then absorbed into the cells in the form of Fe³⁺ [41] and subsequently transported from the roots to the leaves and seeds through the xylem during the growth of Triticum aestivum L.

4.2. Mechanism of Iron Accumulation in Various Parts of Triticum aestivum L. under Iron Deficiency Stress Conditions

This pot planting experiment, conducted under varying soil iron concentrations, revealed that the element contents in various parts of Triticum aestivum L. do not show a linear relationship with the background gradient. Particularly at the maturity stage, the iron content in various Triticum aestivum L. organs generally displayed an initial increase followed by a decrease as the soil iron content varied in the sequence of deficient, moderately deficient, medium, moderately adequate, and adequate. The iron content in stems, leaves, and seeds reached its peak at the concentration gradient of 7.2 mg/kg, with the soil containing 4.5 mg/kg and 7.2 mg/kg considered deficient and moderately deficient, respectively.

Compared to other concentration gradients, the iron deficiency stress environment accelerates the secretion of plant high iron transporters (MAs) from roots to the rhizosphere and activates insoluble iron. This releases a large amount of plant high iron transporters—mugineic acids (MAs), thereby increasing the availability of Fe in the rhizosphere and facilitating plants to directly absorb the high Fe transporter complex, i.e., Fe³⁺-PS [22,42,43]. This is achieved through the specific absorption system located on the root plasma membrane, resulting in an increase in iron content in Triticum aestivum L. roots, stems, and seeds. Likewise, in a stress environment, when the soil’s iron supply is relatively sufficient, it is easier for plant organs to absorb iron, resulting in the highest iron content in all parts of Triticum aestivum L. at a concentration of 7.2 mg/kg. However, when the iron content in the soil is moderate or abundant, the effectiveness of iron in the rhizosphere decreases, thus inhibiting the iron uptake by plants to some extent, leading to a decrease in the iron content in various plant organs.

4.3. Transport and Enrichment of Iron in Triticum aestivum L.

The average transport coefficients of iron in the jointing stage and mature stage of Triticum aestivum L. were 0.316 and 0.411, respectively, both of which were less than 1. This suggests that the transport capacity of iron in the soil by different parts of the Triticum aestivum L. plant is low. As Triticum aestivum L. grows, its transport capacity generally increases. Specifically, at a concentration gradient of 7.2 mg/kg, the translocation factor from underground to aboveground is the highest during the jointing stage, and the enrichment coefficient of roots and stems corresponding to this concentration gradient is the highest at maturity. Combined with the knowledge obtained by the aforementioned studies on the mechanism of iron accumulation in Triticum aestivum L. under iron deficiency stress, the authors suggest that maintaining an iron-deficient soil environment is beneficial for the growth of Triticum aestivum L. Previous studies have indicated that the application of iron fertilizer can alleviate iron deficiency symptoms in plants. However, it has been observed that applying iron fertilizer to plant roots or leaves can rapidly desensitize the plant roots’ response to iron deficiency stress [44,45]. Therefore, this experiment holds significant reference value for determining the amount of iron fertilizer to be applied to the roots. Given that this experiment was conducted in a highly alkaline soil background, the concentration level of the added iron ion solution should be determined by studying iron transport in various soil backgrounds.

Chlorosis, resulting from iron imbalance in calcareous soil, is widespread in northwest and north China. It affects trees, fruit trees, pastures, vegetables, and field crops, with fruit trees being especially vulnerable to chlorosis compared to annual crops, resulting in a significant decline in fruit product quality. The application of iron micro-fertilizer is a rapid and effective method to alleviate symptoms of iron deficiency in plants. The type of iron fertilizer and its application mode can significantly impact the utilization efficiency of iron in plants. This study can offer valuable insights into enhancing plant iron nutrition by promoting efficient iron uptake and transport through regulatory measures, such as fertilization methods and planting patterns.

5. Conclusions

During both the jointing and maturity stages of Triticum aestivum L., there is a preferential enrichment of iron in the underground parts. As Triticum aestivum L. undergoes growth, iron ions in the soil establish coordination bonds with carboxyl and hydroxyl groups. Fe³⁺ is continuously transported from the roots to the leaves and seeds via the xylem controlled by the process of root reduction, which facilitates the migration of iron between the soil and Triticum aestivum L. At maturity, the iron content in various Triticum aestivum L. organs demonstrates a general pattern of initial increase followed by a decrease. This corresponds to the variations in soil iron content categorized as deficient, moderately deficient, medium, moderately adequate, and adequate. This phenomenon can be attributed to the accelerated secretion of high-iron transporter from the Triticum aestivum L. root rhizosphere and the enhanced availability of iron in the rhizosphere under conditions of iron deficiency stress. Additionally, the adjustment of background iron content in the soil has minimal influence on the iron content of Triticum aestivum L. seeds. The conducted experiment reveals that a concentration gradient of 7.2 mg/kg is most favorable for the enrichment of iron in the shoot of Triticum aestivum L. plants. These findings provide valuable insights for developing fertilization strategies aimed at alleviating iron deficiency symptoms in plants growing in similar acidic and alkaline soil conditions.