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14 November 2025

Effect of Reduced Iron Chelate Fertilization on Photosynthesis, Stress Parameters, and Yield of Mandarin Trees

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Group of Fruit Tree Biotechnology, Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Campus de Espinardo, 30100 Murcia, Spain
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Agronomy2025, 15(11), 2622;https://doi.org/10.3390/agronomy15112622
This article belongs to the Section Soil and Plant Nutrition
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Abstract

The objective of this work was to analyze the effect of reducing Fe chelate fertilization (50% and 25% reduction) on soil nutrient content and on different physiological and biochemical parameters in mandarin leaves. The reduction in Fe fertilization efficiently decreased soil Fe content, even in the short-term, without affecting leaf Fe or chlorophyll contents. Reduced iron fertilization increased the accumulation of certain heavy metals in mandarin roots, indicating potential implications for phytoremediation. It is well-established that disturbances of foliar Fe homeostasis can impair the photosynthetic process. Nevertheless, reduction in Fe supply did not negatively affect photosynthetic performance (based on chlorophyll fluorescence parameters), nor did it influence the stress levels of the trees, as indicated by lipid peroxidation. In addition, reduced iron fertilization did not alter peroxidase activity, which is considered a biochemical marker of Fe nutrition in plants. Finally, mandarin production was evaluated over two consecutive years, with no significant variations among the different iron treatments, whereas only minor differences were observed in fruit quality. Overall, these results indicate that reducing Fe fertilization is a feasible strategy, as it does not adversely affect the physiological processes or yield of mandarin trees. Furthermore, this agricultural practice can enhance nutrient use efficiency, thereby contributing to the reduction in soil and aquifer contamination while providing economic benefits to farmers.

1. Introduction

Iron (Fe) plays a crucial role as a micronutrient for plants, contributing significantly to their metabolic processes. About 80–90% of cellular iron is located in chloroplasts. Therefore, any disruption in the foliar iron balance negatively affects chlorophyll biosynthesis and the photosynthetic apparatus [], ultimately impairing plant growth and reducing crop yield [,]. Iron is recognized as one of the most abundant elements in the environment; however, it poses a challenge as a limiting nutrient for plants due to its low solubility, particularly in calcareous and alkaline soils []. Fe is involved in DNA synthesis, energy production and conversion, and nitrogen reduction []. Iron is commonly encountered in two oxidation states: ferric (Fe3+) and ferrous (Fe2+), the latter being the form absorbed by roots. The oxidation state of Fe2+ is easily altered, enabling it to engage in numerous cellular processes. Nevertheless, fine-tuned regulation of this redox state is essential to prevent cellular toxicity []. Iron plays a dual role in the production and removal of reactive oxygen species (ROS). In the presence of molecular oxygen, many low-molecular-weight iron chelates, notably free iron in the Fe3+ or Fe2+ states, can lead to the generation of ROS such as superoxide (O2•−) and hydroxyl radicals (•OH) []. In contrast, iron is also a component of important antioxidant enzymes such as catalases, peroxidases, ascorbate peroxidase, and Fe-superoxide dismutase, which are involved in the control of ROS (O2•− and H2O2) [,], thereby contributing to cellular defense against oxidative stress. Enzymes of this nature play key roles in plant metabolism, including electron transport, redox reactions, and metabolic pathways that are fundamental to plant growth and development []. Under Fe-deficient conditions, the activity of Fe-containing antioxidant enzymes decreases [,,]; therefore, these enzymes are considered reliable biochemical markers of plant Fe status.
An imbalance of iron, whether due to deficiency or excess, interferes with plant metabolism and causes physiological disorders because Fe is a cofactor for many redox enzymes involved in the production of specific hormones, chlorophyll synthesis, and the electron transport chains of chloroplasts and mitochondria [,]. In citrus trees, the symptom of Fe deficiency is commonly known as “iron chlorosis” or “lime-induced chlorosis” when it occurs in calcareous soils. These symptoms are typically observed in young leaves, which turn light yellow to whitish in color, with the veins remaining greener than the rest of the leaf. As Fe deficiency worsens, the volume of the tree canopy decreases, leading to reduced fruit set and yield. Furthermore, the fruits may become smaller, with lower levels of soluble solids and acidity in the juice [].
In order to enhance agricultural productivity, iron is frequently incorporated into fertilizers. However, excessive soil Fe can cause toxicity, especially in flooded soils that favor the presence of reduced, soluble Fe2+ []. Furthermore, excessive iron in the soil solution may interfere with the absorption of other micronutrients, thereby affecting chlorophyll biosynthesis, the overall photosynthetic process, and plant development. Consequently, this can lead to a reduction in CO2 assimilation and a shortage of sugars that are crucial for the plant’s survival and for the storage of reserve carbohydrates in sink organs [].
The soils in Murcia (Southeast Spain) are characterized by their limestone composition, high calcium carbonate content, and alkaline pH []. Iron chlorosis in citrus is frequently associated with cultivation in calcareous soils, with Fe deficiency being the most common micronutrient deficiency in calcareous soils []. This leads farmers to apply large quantities of Fe chelates to facilitate iron uptake by plants. Fe (o,o-EDDHA) is one of the most effective Fe fertilizers used in calcareous soils to avoid iron deficiency in fruit crops, especially in citrus plants []. Unfortunately, this agricultural management has been shown to have adverse effects on plants and contribute to soil and aquifer contamination. In this context, the objective of this work was to study how the reduction in Fe chelate fertilization may affect mandarin trees’ physiology and fruit productivity in a commercial orchard located in Murcia.

2. Materials and Methods

Field trials were conducted in a commercial orchard located in Pozo Estrecho 37.70116° N, 0.99216° W, Spain). Nadorcott mandarin (Citrus reticulata) trees cv., grafted on ‘Citrus macrophylla’ rootstock and cultivated in clay soil characterized by a low organic C content and a high CaCO3 concentration (pH 8–8.2), were studied. At the beginning of the experiments, trees were 4 years old, and planted at a density of 417 trees/hectare.
The proposed crop management treatments, in comparison to the fertilization method employed by the company (control treatment, 29.1 Kg Fe ha−1 year−1 as RUCK-Fe, Ruraltech, Murcia, Spain), included: 75% Fe (21.8 kg Fe ha−1 year−1) and 50% Fe (14.6 kg Fe ha−1 year−1). Treatments started in May 2023. Later, three different samplings were performed after 4 months (September 2023, short-term, hereinafter ST), 9 months (February 2024, mid-term, MT), and 20 months of the treatment (October 2024, long-term, LT). Fully expanded, non-senescent leaves were used for biochemical analyses. Leaf samples collected in the field were frozen in a cryogenic container (VOYAGEUR PLUS, Air Liquide, Marne La Vallée, France ) containing liquid nitrogen and stored at −80 °C. Approximately, two grams of small fibrous roots (≤2 mm in diameter) were collected, kept on ice during transport, and then frozen at −20 °C. Soil samples were collected using an auger (5 cm diameter) at a depth of 20–30 cm.

2.1. Mineral Nutrient Content

Plant material (leaves and roots) was thoroughly washed first with tap water and then three times with distilled water. The plant material was oven-dried at 60 °C for 4 days, whereas soil samples were air-dried for 48 h. The dried material was finely ground, and the leaf, root, and soil samples were sieved. Subsequently, samples were subjected to an acid digestion using a high-performance microwave in the presence of HNO3/H2O2 (4:1, v/v) (Ultraclave, Milestone, Shelton, CT, USA). The determination of cation contents in the soil, leaves, and roots was conducted by inductively coupled plasma-optical emission spectroscopy (ICP-OES, iCAP 6000SERIES spectrometer, Thermo Scientific, Madrid, Spain) at the Ionomics Service from CEBAS-CSIC (Murcia, Spain).

2.2. Optical Determination of Chlorophylls, Flavonols and Anthocyanins

Chlorophyll and flavonol contents, as well as the nitrogen balance index (NBI index), were measured using a portable optical leaf-clip meter DUALEX® (FORCE-A, Orsay, France). Measurements were performed between 9:00 and 10:00 h (GMT).

2.3. Chlorophyll Fluorescence Determination

Chlorophyll fluorescence parameters in mandarin leaves were determined using a portable FSM fluorimeter (Hansatech Instruments Ltd., Pentney, UK). Measurements were carried out between 9:00 and 11:00 h (GMT) in dark-adapted leaves. The different chlorophyll fluorescence parameters were obtained according to [].

2.4. Lipid Peroxidation and Peroxidase Activity Determinations

The levels of lipid peroxidation in mandarin leaves were analyzed as an indicator of oxidative stress. Mandarin leaves (0.2 g) were ground in liquid nitrogen to a fine powder, followed by extraction with 1 M perchloric acid. Thiobarbituric reactive substances (nmol TBARS g−1 FW) were determined in the extract following the procedure described by [].
For the measurement of peroxidase (POX) activity, leaf samples were ground with liquid nitrogen and extracted with 50 mM Tris-acetate buffer (pH 6.0) containing 0.1 mM EDTA and 0.2% Triton X-100 (Sigma-Aldrich, Madrid, Spain). Samples were centrifuged at 10,000 rpm for 10 min at 4 °C. Supernatants were desalted through Shephadex G-25 columns (Fisher Scientific, Madrid, Spain), and POX activity was spectrophotometrically determined in a reaction mix containing 50 mM Tris-acetate buffer (pH 5.0) and 0.5 mM H2O2, using 1.0 mM 4-methoxy-α-naphtol (ε595 = 21,600 M−1 cm−1) as the electron donor [].

2.5. Fruit Yield and Quality

Fruit yield was evaluated based on the commercial harvest from five different trees per treatment, taken after 8 months (January 2024) and 20 months (January 2025, long-term treatment) since the beginning of the experiment. The number of fruits as well as the fruit production per tree (Kg tree−1) were determined. To test fruit quality, soluble solids (°Brix) and acidity were determined using a portable PAL-BX/ACID refractometer (Atago Co. Ltd., Tokyo, Japan).

2.6. Statistical Analysis

Data were subjected to analysis of variance (ANOVA), followed (when applicable) by Tukey’s Multiple Range Test (p ≤ 0.05), using SPSS® software (IBM SPSS Statistics, version 27.0.0.0, Armonk, NY, USA).

3. Results

3.1. Effect of Agronomic Management on Nutrient Contents

Reducing iron fertilization effectively decreased its accumulation in the soil even in the short-term (ST). In this sense, a significant decrease in the Fe content in soil was observed in the 75% Fe and 50% Fe treatments, with decreases of 13% and 21%, respectively (Figure 1a). A similar effect was observed in the mid-term (MT), with significant decreases in soil iron of 20% and 25% with the 75% Fe and 50% Fe treatments, respectively. In the long-term (LT), a progressive decrease in Fe concentration in soil was observed, but the differences were statistically significant only in the 50% Fe treatment (Figure 1a).
Figure 1. Effect of the agronomic management of iron fertilization on Fe contents in soil (a), roots (b), and leaves (c) from mandarin trees in the short-term (ST), mid-term (MT). and long-term (LT). The values correspond to the mean ± SE of 5 different biological samples. Different letters, in the same type of column, indicate significant differences according to Tukey’s Test (p < 0.05). No letters indicate no significant differences (p > 0.05).
Regarding roots, in ST, the 75% Fe treatment did not reduce the Fe levels significantly whereas the 50% Fe treatment displayed higher levels than those of the control (Figure 1b). However, the root Fe content did not vary between treatments at MT and LT. Concerning leaves, Fe levels did not show significant changes between the different treatments in MT and LT (Figure 1c). Only a significant decline in leaf Fe levels was observed at ST in the 50% Fe treatment, however, the leaf Fe content remained within the optimal range for citrus [,].
Short-term Fe fertilization reduction increased the root cation concentration in the 50% Fe treatment (Table 1). In this regard, higher contents in Cd, Cu, Mn, Mg, Na, S, and Zn were observed, with increases ranging from 1.4- to 5-fold (Table 1).
Table 1. Effect of short-term addition on the level of selected cations in mandarin roots. Values represent the mean ± SE of five independent biological replicates. Different letters within the same column indicate significant differences according to Tukey’s test (p < 0.05). aF-values significant at 99.9% (***) or 95% (*) levels of probability. No letters indicate no significant differences (p > 0.05). Data expressed as mg kg−1 DW.
At MT, the 50% Fe treatment led to an increase in the levels of certain cations within the roots. This resulted in a greater content of Cu, Mg, Zn, and Cd (Table 2). Specifically, the Cu levels doubled, while the increases in root concentrations of Mg and Zn were 1.5-fold and 1.7-fold, respectively (Table 2). Furthermore, in the mid-term, a significant accumulation of Cd, which was four times higher, was observed in the roots of trees subjected to a 50% Fe supply. Additionally, a threefold rise in Cr content was also recorded (Table 2).
Table 2. Effect of mid-term agronomic management of iron fertilization on the level of selected cations in mandarin roots. Values represent the mean ± SE of five independent biological replicates. Different letters within the same column indicate significant differences according to Tukey’s test (p < 0.05). aF-values significant at 99.9% (***), 99% (**), or 95% (*) levels of probability. For more details, please see Table 1. No letters indicate no significant differences (p > 0.05). Data expressed as mg kg−1 DW.
At long-term, an increase was also observed in the contents of some cations in roots by the effect of Fe supply reduction. The 50% iron reduction increased the root Cu content by 2.3-fold and the Zn levels showed a 3-fold increase (Table 3). Concerning heavy metals, 50% Fe treatment increased the uptake of heavy metals, but in this case, only Cr changes were statistically significant (Table 3).
Table 3. Effect of long-term agronomic management of iron fertilization in the level of selected cations in mandarin roots. Values represent the mean ± SE of five independent biological replicates. Different letters within the same column indicate significant differences according to Tukey’s test (p < 0.05). aF-values significant at 95% (*) levels of probability. No letters indicate no significant differences (p > 0.05). Data expressed as mg kg−1 DW.
Notably, significant Na levels were detected in roots over the long-term (Table 3). This may be attributed to the partial use of “poor quality water” containing salts (>5 dS/m EC) during the June–September period each year. Nonetheless, mandarin trees can effectively exclude Na from their leaves by accumulating it in the roots. In this context, over the long-term, the Na content in leaves only varied between 700 and 850 ppm (data not showed) in comparison to the highest Na levels recorded in the roots (see Table 3).

3.2. Effect of Agronomic Management on Pigment Contents and Chlorophyll Fluorescence

Measurements obtained with the Dualex Scientific device showed no significant effects of the treatments on chlorophyll, flavonol contents, or on the nitrogen balance index (NBI).
The measurement of chlorophyll fluorescence provides a means to gain knowledge about the efficiency of photochemical and heat dissipation processes in the chloroplast []. No adverse effects on photosynthesis were observed in mandarin trees subjected to reduced Fe fertilization. The Fv/Fm parameter, which provides information regarding PSII efficiency, did not show significant changes in either the ST or LT assessments (Figure 2a). Additionally, a significant increase was noted at MT with the 75% Fe treatment (Figure 2a). It is noteworthy that the Fv/Fm values were lower at MT compared to other sampling periods, with values ranging from 0.6 to 0.7. On the other hand, Fv/Fm data were above 0.8 for ST and LT, which indicates a good photosynthetic performance []. These results were parallel to those for the quantum yield parameter [Y(II)], which did not undergo significant changes in most cases, but even increased significantly in the MT with 75% Fe treatment (Figure 2b). Similarly to the lower Fv/Fm values detected at MT, motivated by the decrease in temperatures, the Y(II) values were also lower in relation to the data detected in ST and LT when the temperature values were higher. In contrast, no significant changes were detected in the parameter qP, with the data recorded for this parameter similar among sampling periods (Figure 2c). Interestingly, ETR data were always higher in mandarin leaves subjected to Fe fertilization reduction (Figure 2d).
Figure 2. Effect of reduced iron fertilization on photosynthetic and photochemical parameters in mandarin leaves at the short-term (ST), mid-term (MT), and long-term (LT) evaluations. (a) Fv/Fm, (b) qP, (c) Y(II), and (d) ETR. The values correspond to the mean ± SE (n = 6 independent biological replicates). Different letters within each sampling period indicate significant differences according to Tukey’s test (p < 0.05). No letters indicate no significant differences (p > 0.05).
On the other hand, at ST, both NPQ and qN were statistically higher in the 50% Fe treatment, as well as NPQ at S3 in trees subjected to 75% Fe (Figure 3).
Figure 3. Effect of reduced iron fertilization on non-photochemical quenching parameters in mandarin leaves at the short-term (ST), medium-term (MT), and long-term (LT) evaluations. (a) NPQ and (b) qNP. The values correspond to the mean ± SE (n = 6 independent biological replicates). Different letters within each sampling period indicate significant differences according to Tukey’s test (p < 0.05). No letters indicate no significant differences (p > 0.05).

3.3. Lipid Peroxidation and Peroxidase Activity Determinations

The extent of lipid peroxidation, monitored as TBARS, was assessed in mandarin leaves as an oxidative stress marker in response to the reduction in Fe fertilization. In general, the reduction in Fe fertilization did not increase the lipid peroxidation levels, and even lower values were observed at ST and MT in the 50% Fe and 75% Fe treatments (Figure 4a), suggesting that the control trees were more stressed that those subjected to the Fe reduction. Finally, at LT, no statistical differences among treatments were observed (Figure 4a).
Figure 4. Effect of the agronomic management of iron fertilization on (a) lipid peroxidation and (b) total peroxidase (POX) activity in mandarin leaves at the short-term (ST), medium-term (MT), and long-term (LT) evaluations. Lipid peroxidation was determined as thiobarbituric acid reactive substances (TBARS). The values correspond to the mean ± SE (n = 6 independent biological replicates). Different letters within each sampling period indicate significant differences according to Tukey’s test (p < 0.05). No letters indicate no significant differences (p > 0.05).
Peroxidases (EC 1.11.1.7) are Fe-containing heme proteins that catalyze the univalent oxidation of a wide range of organic and inorganic substrates at the expense of H2O2 []. They can serve as a biochemical marker of Fe nutrition in plants. No significant variations in POX activity were observed among samples at any of the three sampling times (Figure 4b).

3.4. Fruit Production and Quality

After 8 months of the beginning of the treatments, fruit yield and quality were evaluated. Production, measured as kg mandarins tree−1, was not significantly affected by the treatments (Figure 5). Minor differences were observed concerning fruit quality. In the first year (2024), only 50% Fe treatment exhibited a significant decrease of °Brix in relation to control trees (Figure 6a), with no changes in acidity (Figure 6b). Likewise, in the second year (2025), Fe fertilization had no significant effect on fruit production; however, there was a reduction in mandarin production relative to the first year (Figure 5). The increased Na accumulation detected in the roots may have had a detrimental effect on mandarin production during this second year. Regarding fruit quality, no differences in °Brix were recorded in the second year (Figure 6a), but significant differences in acidity were observed between the Fe treatments, with fruits from the 50% Fe treatment displaying higher acidity than those from the 75% Fe treatment (Figure 6b). Overall, these findings suggest that reducing the iron content does not have a substantial impact on the quality of the fruit.
Figure 5. Effect of agronomic management of iron fertilization on mandarin yield in 2024 (black columns) and 2025 (grey columns). Fruit production per tree (kg mandarins/tree) was calculated using five trees per treatment. No statistically significant differences were detected among treatments according to Tukey’s test (p < 0.05).
Figure 6. Effect of decreased iron nutrition on mandarin quality in the years 2024 (black columns) and 2025 (grey columns). (a) °Brix and (b) acidity. The values correspond to the mean ± SE of 25 different fruits per treatment and year. Different letters within each sampling period indicate significant differences according to Tukey’s test (p < 0.05). No letters indicate no significant differences (p > 0.05).

4. Discussion

Iron serves as a vital micronutrient for plants, significantly contributing to numerous metabolic functions []. Iron plays a crucial role in essential processes within plant cells, including photosynthesis and respiration, and it also contributes to the defense against ROS by being a component of various antioxidant enzymes, such as catalases, peroxidases, and Fe-containing superoxide dismutase []. While abundant in soil, iron is often in a form that is not readily available for plant uptake. This situation leads farmers to use large quantities of Fe-chelates, which could cause a negative effect on plants, as well as soil and aquifer contamination when applied in excess. Consequently, our primary objective was to lower the quantity of Fe-chelates utilized by farmers while examining the response of mandarin trees over time regarding pigment content, photosynthesis, leaf stress levels, POX activity as a biochemical indicator of Fe levels in leaves, and ultimately, yield and quality of the fruits.
As far as we know, the information about the effect of effect of reduced iron chelate fertilization on photosynthesis, stress parameters, and yield citrus trees is very limited. However, we identified a study in which the authors partially replaced the synthetic chelate Fe (o,o-EDDHA) with a commercially available humic substance in calcareous soils from southeastern Spain (Murcia). The findings revealed that reducing the amount of Fe chelate applied could yield both economic advantages and potential ecological benefits [].
The reduced Fe fertilization was reflected in a decrease of this micronutrient in soil, even at short-term. It is well-known that an excess of iron in the soil can be detrimental to plant health, soil ecology, and nearby aquifers. While iron is an essential micronutrient, its presence at high levels, especially under anaerobic or water-logged conditions, can be detrimental for plants. In soils with excess iron, plant roots can enhance their absorption of iron, typically by converting Fe3+ to Fe2+, a process that may also involve the release of ROS []. The root is the first organ to sense excess soil Fe, and Fe toxicity plays a direct role in the modulation of root system architecture []. In Arabidopsis plants, excess iron Fe affects root tip zone growth through the overaccumulation of nitric oxide []. In addition, Fe excess can increase H2O2 production in the tip zone and arrest primary root growth [].
Excess soil iron can also be linked to water contamination. Iron can leach into groundwater or surface waters, especially in acidic or poorly drained soils, contributing to pollution []. Moreover, iron can also bind with phosphorus in sediments and release it under anaerobic conditions, contributing to algal blooms [].
Interestingly, the decrease in Fe fertilization by 50% produced an increase in the content of some macronutrients and micronutrients in roots, especially Cu and Zn. It is likely that a lower concentration of iron in the rhizosphere zone may promote the absorption of alternative cations. In that regard, it has been reported that Fe can compete with other transition metals such as Mn, Cu, or Zn in uptake, transport, or even chemical reactions in plant cells []. High concentrations of Fe in soil and around rhizosphere can also produce indirect damage by affecting the proper absorption of other essential metals. In fact, deficiency or excess of any nutrient can cause an imbalance in other nutrients for uptake because of their interactions. Moreover, high levels of Fe in the soil solution may result in its precipitation over the roots, forming a crust of ferric oxide that alters the absorption of other nutrients like Zn and Mn [,].. These findings are consistent with our experimental observation on the mineral nutrition of roots. Fe nutrition also affects S transport in plants, with Fe starvation increasing the expression of sulfate transporters, which enhances S uptake from the soil [], whereas S concentration was lower in plant shoots under excess iron []. In addition, the increase in the content of some heavy metals in roots was also noticed, which means that reduction in Fe supply, in addition to reducing soil contamination with excess Fe, may have implications for phytoremediation; further studies are needed to assess translocation to shoots/fruits and total metal removal per hectare.
Chlorophyll biosynthesis is dependent on Fe for the formation of precursors of the chlorophyll molecules []. It is well-known that Fe deficiency leads to a decline in chlorophyll contents, resulting in leaf chlorosis. It has been described that both severe and extreme iron deficiency reduced the chlorophyll contents in different plant species in comparison to the control plants’ growth with optimal Fe levels, which correlated with decreases in the leaf Fe contents [,,]. Therefore, it could be hypothesized that a reduction in iron supplementation during the fertilization of mandarin trees might negatively affect chlorophyll content. However, under our experimental conditions, the lower Fe input had no significant effects in the leaf Fe content (see Figure 1), nor in the chlorophyll levels (p values 0.186, 0.755, and 0.409 for ST, MT, and LT experiments, respectively). In this sense, the content of Fe in leaves ranged from 60 to 100 mg kg−1 DW, with no changes between the different treatments, these values being considered as normal in Citrus plants [,,]. This observation could also be explained by the fact that in periods of low external Fe supply, the tree may remobilize stored Fe to young leaves, preventing visible deficiency symptoms [].
Disturbance of foliar Fe homeostasis leads to the impaired biosynthesis of chlorophylls and photosynthetic machinery disorders [], strongly affecting the photosynthesis process []. It is noteworthy that the reduction in Fe fertilization did not negatively influence the photosynthesis process, according to the chlorophyll fluorescence measurements. Fv/Fm provides information on the functioning of PSII, while Y(PSII) indicates the amount of light energy absorbed by PSII and used in photochemical processes []. In contrast, non-photochemical quenching parameters illustrate the plants’ capability to cope with environmental stress. An increase in these parameters denotes the safe dissipation of excess light energy as heat, acting as a defense mechanism for chloroplasts under stress conditions []. Generally, leaves that are deficient in Fe show a lower photosynthetic rate, which is often associated with a decline in photosynthetic electron transport, Y(II) and qP parameters, as well as with increased NPQ [,]. The results obtained from chlorophyll fluorescence determinations are particularly notable, as the decrease in Fe supply led to improved photosynthesis performance in mandarin trees. For instance, at MT, the 75% Fe treatment led to an increase in the photochemical quenching parameters Fv/Fm and Y(PSII). It is noteworthy that the Fv/Fm values were lower at MT compared to other sampling periods, with values ranging from 0.6 to 0.7. This may be due to the lower temperatures recorded for MT (12.0 °C average temperature for February 2024) than for ST (23.6 °C for September 2023) and LT (20.0 °C for October 2024). These temperature variations can influence the photosynthesis process, as a decrease in temperature may lead to the photoinhibition of PSII to prevent damage to the chloroplast, as indicated by the Fv/Fm data. These data correlated with lower leaf Fe contents detected at MT relative to the ST and LT samples. It can be possible that lower temperatures could affect Fe uptake and transport to the leaves. In fact, it has been reported that leaf and shoot Fe content increased linearly with soil temperature in citrus plants []. Furthermore, it has been reported that citrus leaves exhibit seasonal variations in nutrient concentrations [].
Moreover, at short- and long-term, the 50% Fe treatment exhibited an increase in the non-photochemical quenching parameters NPQ and qNP, whereas the photochemical quenching parameters were not affected. Increases in these parameters suggest enhanced photoprotective energy dissipation, which may help maintain photochemical performance under altered Fe supply. In different plant species, including sugar beet, pear, and peach, a strong NPQ increase in response to Fe deficiency was reported, in parallel with significant decreases in the photochemical quenching parameters Y(II) and qP [].
In addition, it is worthy to mention that in this study the decrease in Fe fertilization did not negatively impact ETR. This is of interest, taking into account that Fe is an essential component of various photosynthetic electron transporters in both PSI and PSII, actively participating in this process during photosynthesis []. In thylakoid membranes, approximately 20 Fe atoms are directly involved in the electron transport chain. The elevated Fe requirements in thylakoids, along with its role in chlorophyll biosynthesis, can explain the high sensitivity of chloroplasts to Fe deficiency []. Thus, the current study’s observations that a reduction of up to 50% in Fe fertilization has no negative effect on either leaf Fe content or ETR suggests that an excess of Fe fertilization is routinely applied on commercial mandarin orchards.
The different iron management strategies did not influence the stress levels of the trees. In this sense, the lipid peroxidation levels decreased with reduced Fe fertilization in relation to traditional farming treatments, particularly in the mid-term. It has been reported that excess Fe2+ induces the formation of free radicals in several plant species []. The elevated lipid peroxidation values monitored in control mandarin leaves may suggest that the Fe input normally provided by the farmer could induce a mild oxidative stress. Indeed, Fe is a component of the lipoxygenase enzyme, which facilitates the peroxidation of linolenic and linoleic acids []. Although we did not assess lipoxygenase activity, it can be inferred that the control plants might exhibit greater lipoxygenase activity compared to the other treatments, thereby leading to the increased lipid peroxidation values. Moreover, [] provided evidence linking excess iron, introduced via nanoparticle or ionic forms, and elevated lipid peroxidation in Citrus maxima; this was also associated with the activation of antioxidant enzymes, suggesting plant adaptation to excess iron.
Iron is a component of important heme enzymes []. Thus, iron-dependent enzymes can be effective biochemical markers for determining the leaf Fe concentration. Accordingly, plant peroxidases may function as biochemical markers for the availability of iron in plants since iron deficiency results in diminished peroxidase activity [], while an excess of iron can lead to an increase in its activity [] as a result of the oxidative stress generated in these situations. This effect is especially negative under water shortage conditions because it has been described that the damage produced in the photosynthetic tissue is caused by Fe-catalyzed ROS formation in the chloroplasts []. Overall, and in parallel to the observed data on the Fe and chlorophyll contents in leaves or in chlorophyll fluorescence, no significant changes were observed in the total POX activity in mandarin leaves.

5. Conclusions

Moderate reductions in Fe fertilization (up to 50%) did not significantly affect photosynthesis, Fe content, or yield in the short-term, suggesting that current Fe inputs might be higher than necessary. Long-term studies including environmental and economic evaluations would be required to confirm these potential benefits.

Author Contributions

Conceptualization, P.D.-V. and J.A.H.; methodology, A.H.-C., C.J.-M., P.D.-V. and J.A.H.; validation, P.D.-V. and J.A.H.; formal analysis, P.D.-V. and J.A.H.; investigation, A.H.-C., C.J.-M., P.D.-V. and J.A.H.; resources, P.D.-V. and J.A.H.; data curation, P.D.-V. and J.A.H.; writing—original draft preparation, P.D.-V. and J.A.H.; writing—review and editing, P.D.-V. and J.A.H.; visualization, P.D.-V. and J.A.H.; supervision, P.D.-V. and J.A.H.; project administration, J.A.H.; funding acquisition, J.A.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Center for Technological Development and Innovation (CDTI, SPAIN) through the company Roymaga S.L. (Spain): IDI-20221056.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We would like to sincerely thank Patricia Molina for all her help and dedication in managing the mandarin trees throughout the duration of the project.

Conflicts of Interest

The authors declare no conflicts of interest.

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Research Progress on the Regulation of Plant Rhizosphere Oxygen Environment by Micro-Nano Bubbles and Their Application Prospects in Alleviating Hypoxic Stress
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