Physiological Responses to Fe Deficiency in Split-Root Tomato Plants: Possible Roles of Auxin and Ethylene?

Abstract

Iron (Fe) bioavailability in soils is often limited and can be further exacerbated by a non- homogeneous distribution in the soil profile, which has been demonstrated to vary both in space and time. Consequently, plants respond with morphological and physiological modifications at the root level involving a complex local and systemic signaling machinery. The present work unravels the role of two phytohormones (i.e., ethylene and auxin) and their integrated signaling in plant response to Fe deficiency. Inhibitors of auxin polar transport and of ethylene biosynthesis (N-1-naphthylphthalamic acid - NPA and aminoethoxyvinylglycine - AVG, respectively) were applied on tomato (Solanum lycopersicum L.) plants grown by the split-root technique, which allows to simulate condition of Fe heterogeneous distribution. Results showed that plants, exposed to an uneven Fe supply, triggered a complex auxin-ethylene signaling. A systemic action of auxin on FERRIC REDUCTASE OXIDASE 1 (SlFRO1) expression was revealed, while ethylene signaling was effective both locally and systemically. In addition, the investigation of Fe concentration in tissues showed that when leaves overcame Fe deficiency a Fe “steady state” was maintained. Therefore, physiological adaptation to this heterogeneous Fe supply could be mediated by the integration of the complex signaling pathways prompted by both auxin and ethylene activities.

1. Introduction

Mineral elements are crucial for plant growth and productivity, but they are often unevenly distributed within the upper layer of soil as well as along its profile, rather than homogeneously distributed. In particular, soil is characterized by areas defined as “nutrient hotspots”, in which nutrients are more abundant [1,2,3]. Thus, in the natural growth conditions, plants are often exposed to a patchy distribution of nutrients [4,5], which may affect different aspects of plant growth, function and productivity with direct consequences on nutrient uptake and root growth and development. Therefore, understanding the responses of plants to soil heterogeneity is critical in order to develop strategies to optimize plant production.

Extensive research analyzing how plants cope with uneven nutrient availabilities in the soil has been previously performed [6,7,8]. It is well known that nutrient hotspots could likely increase the nutrient mass flux (nutrient movement via soil water driven by plant transpiration) to the root surface [9], and determine a tropic root growth towards the nutrient-rich areas [10]. Indeed, plants are able to efficiently exploit nutrient-rich patches by selectively increasing root growth proliferation [11,12]. However, nutrient uptake is highly affected not only by nutrient concentration, but also by its chemical form determining the available fraction for plant uptake. In particular, the nutrient bioavailability is influenced by physical, chemical, and biological proprieties of soil [13,14]. Iron (Fe) availability, for instance, depends primarily on the soil pH and redox potential [15]. Iron is the fourth most abundant element on the Earth’s crust, but it is poorly available to plants, especially in alkaline soils [16]. The imbalance between Fe solubility in soils and the demand of Fe by the plant is the primary cause of leaf Fe chlorosis [17]. Iron plays a key role in crucial metabolic processes such as photosynthesis, respiration, and nitrogen assimilation, other than chlorophyll synthesis [18,19,20]. Being one of the main constraints for plant growth and productivity, Fe nutrition has been widely investigated with the aim of improving the nutrient use efficiency, and thus increasing agricultural productivity. Thus, a better exploitation of soil endogenous resources might lead to a reduction in the external Fe fertilizer applications, also resulting in the reduction of the environmental issues posed by such agronomic practices [21].

When plants are challenged with Fe limitation a reduction-based strategy, known as Strategy I (all the dicots and non-graminaceous monocots), or a chelation-based strategy, known as Strategy II (only monocots), allows to improve Fe mobilization and uptake by roots [22]. Tomato (Solanum lycopersicum L.), as Strategy I species, acidifies the rhizosphere through plasma membrane H⁺-ATPase activity to solubilize Fe³⁺, which is reduced to Fe²⁺ by the Fe³⁺-chelate reductase (FCR) activity, and the resulting Fe²⁺ is transported into the root cells via the specific Fe transporter (IRT1) [23,24]. Physiological responses [25,26] are also associated with root morphological changes, including enhanced formation of subapical root hairs, transfer cells, and root branching [27,28,29]. Finally, various studies have highlighted that the regulation of Strategy I plant responses to Fe stress is mediated by phytohormones [30,31], by which Fe deficiency signals could be transmitted within the plant [30,32,33,34]. It has been suggested that a crucial role is thereby played by both auxin and ethylene, which might interact either synergistically or antagonistically in signaling processes [29,33,34,35,36].

Auxin and ethylene are supposed to be responsible for the generation of a long-distance signal, which is mediated by an unknown factor, moving from shoots to roots [37,38,39], and a local signal, which originates in the roots and directly activates the transcriptional network of FER [31,40]. The major role in the long-distance Fe signaling pathway has been attributed to auxin (indole-3-acetic acid, IAA), since its biosynthesis mainly occurs in aerial tissues (i.e., apical meristems) as well as at level of the root tips [41,42,43]; then, it is rapidly transported systemically from shoots to roots via the polar auxin transport (PAT) through the phloem, or, locally, by the cell-to-cell active transport [44,45]. Thus, auxin could represent the upstream signal in modulating Fe deficiency responses [46].

In the intricate signaling network of Fe availability, auxin does not operate independently, but it communicates Fe demand to the root apparatus and interacts with other hormonal molecules that act locally as enhancers of Fe deficiency responses, such as ethylene [47]. Ethylene is biosynthesized from S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid (ACC) [48,49], and it has been proposed as an essential component in the transduction of Fe deficiency signal, since Fe-deficient roots increase ethylene biosynthesis to induce the well-characterized adaptive changes in root architecture [50,51]. Consistently, plants treated with exogenous ACC, the precursor of ethylene, showed typical morphological changes of Fe deficiency responses [52,53].

Since roots can sense locally the changes in the external medium of Fe supply, the primary perception of Fe deficiency could originate in the roots. Iron deficiency could generate the transmissible information about the presence of nutrients from roots towards the leaves [54]. In addition, Fe from the phloem can act as signal to inhibit the expression of Fe-uptake genes [31,32,55,56].

In some experiments using the split-root system with various Strategy I plants (like tomato, Arabidopsis, and cucumber), it has been found that both expressions of Fe-uptake genes and activity of Fe³⁺-chelate reductase enzyme was increased in the Fe-sufficient half of the root system [38,57,58]. It remains unclear how the signal of Fe deficiency is transmitted from the Fe-deficient to Fe-sufficient half of the root [38,59].

The present work addresses the role of ethylene and auxin and their integrated signaling in plant response to Fe deficiency. Inhibitors of auxin polar transport and of ethylene biosynthesis (N-1-naphthylphthalamic acid - NPA and aminoethoxyvinylglycine - AVG, respectively) were applied on tomato (Solanum lycopersicum L.) plants grown by the split-root technique. This latter technique is a useful tool to demonstrate root-to-shoot and shoot-to-root communication, allowing to discriminate between systemic and localized signals involved in the regulation of Fe deficiency responses. We further used stable labelled Fe isotope (⁵⁷Fe) to reveal the possible effect of Fe on regulating the complex machinery of Fe deficiency signaling and the intricate communication mechanisms between roots and shoots.

2. Materials and Methods

2.1. Plant Growth Conditions and the Split-Root System

Tomato (Solanum lycopersicum L., cv. Marmande) seeds were sown in plastic boxes, containing several layers of wet filter paper, and kept in the dark at 25 °C for 9 days to allow germination [60]. Afterwards, seedlings were selected based on uniformity of size to be transferred in plastic 1.5 L pots (10 seedlings/pot), filled with a continuously aerated nutrient solution (NS), composed as follows (mM): 2 Ca(NO₃)₂, 0.7 K₂SO₄, 0.1 KH₂PO₄, 0.1 KCl, 0.5 MgSO₄, and (µM): 10 H₃BO₃, 0.5 MnSO₄, 0.2 CuSO₄, 0.5 ZnSO₄, 0.01 (NH₄)₆Mo₇O₂₄, and 5 Fe³⁺-EDTA [61].

After 2 weeks of low Fe availability (5 µM Fe³⁺-EDTA), the root apparatus of each plant was divided into two approximately equal parts and separated into two compartments (split-root system); each compartment was filled with 1 L NS, containing different Fe concentrations, i.e., 40 µM Fe³⁺-EDTA or 40 µM ⁵⁷Fe-EDTA for the Fe-sufficient (+Fe) and 0 µM for Fe-deficient (-Fe) condition (Figure 1A). The choice of the experimental cultivation scheme was based on our previous work [62].

Plants were grown hydroponically in a climatic growth chamber with day/night cycle of 14/10 h, temperature regime of 24/19 °C, light intensity of about 250 µmol m⁻² s⁻¹ at plant level, and relative humidity of approximately 70%. The NSs were renewed every 3 days.

2.2. Phytohormonal Inhibitor Treatments and Plant Sampling

The plants were cultured in the split-root system for 1 week in order to induce Fe deficiency response, as shown in Figure 1B, according to our previous work [62]. On the sixth day, plants were treated with N-1-naphthylphthalamic acid (NPA), the inhibitor of the polar transport of auxin, and aminoethoxyvinylglycine (AVG), inhibitor of ethylene synthesis (Figure 1B).

Both NPA and AVG were dissolved in dimethyl sulfoxide (DMSO) and added at the final concentration of 10 and 0.1 µM, respectively [63,64].

At the end of the experiment with 10 µM NPA or 0.1 µM AVG the different conditions were as follows: the 40/0 µM Fe³⁺-EDTA treatment as control; the 40/0 µM Fe³⁺-EDTA with NPA or AVG treatment added to Fe-sufficient part of the container and the 40/0 µM Fe³⁺-EDTA with NPA or AVG treatment added to Fe-deficient part of the container (each side of “/” represents the two different compartments of the same container).

At harvest (30 days after sowing), the Fe³⁺-chelate reductase activity was assayed in vivo on tomato roots. Shoot and root samples were separated, quickly frozen in liquid N₂, and stored at −80 °C for subsequent analysis.

2.3. Determination of Chlorophyll Content

After 7 days of growth in the split-root system, the chlorophyll content per unit area was measured on the youngest fully expanded leaves of each plant, by using a portable chlorophyll meter SPAD-502 (Minolta Co., Osaka, Japan), and recorded values were expressed as “Soil Plant Analysis Development” (SPAD) units.

2.4. Natural and ⁵⁷Fe Accumulation in Shoots and Roots

Uptake of ⁵⁷Fe by split-root tomato plants was determined by measuring the ⁵⁷Fe accumulation in shoots and in both Fe-deficient and Fe-sufficient portions of the root system (Figure 1A). Plants were grown for a week in a NS containing 40 µM ⁵⁷Fe-EDTA (94.87%) (ISC Science, Oviedo, Spain), titrated to pH 6 with 1 M KOH, as previously described [65]. At the end of the treatment with ⁵⁷Fe-EDTA (7 days after application), roots were washed for 15 min in 0.5 mM CaSO₄ to allow the removal of ⁵⁷Fe non-specifically bound to the cell wall [65]. Shoot and root tissues were then oven-dried at 60 °C for 48 h, weighed and then digested with concentrated HNO₃ [65% (v/v), Carlo Erba], using a single reaction chamber (SRC, UltraWAVE, Milestone Inc, Shelton, CT, USA). The mineralized solutions were spiked with 100 ppm ⁵⁴Fe (99.92%) standard solution (ISC Science, Oviedo, Spain) and Fe isotope ratios measurements were carried out by ICP-MS (Agilent 7900, Agilent Technologies, Santa Clara, CA, USA) [66,67,68]. Iron concentration was determined by isotope dilution analysis using a collision cell ICP-MS instrument with helium as collision gas. Instrument and octopole operating condition are summarized in Supplementary Materials Table S1. Isotope Pattern Deconvolution (IPD) is the most complete mathematical approach to perform multiple-spiking Isotopic Dilution (ID) [69,70], needed to quantify natural-abundance element and two enriched tracers [71]. In this work a mixture of natural-abundance Fe and ⁵⁷Fe-enriched Fe was determined by the addition of a second Fe tracer ⁵⁴Fe-enriched.

2.5. Determination of Fe^3+-Chelate Reductase Activity by Intact Roots

The Fe³⁺-chelate reductase activity of tomato roots was assayed colorimetrically, using bathophenantrolinedisulfonate (BPDS) reagent [62,72]. Briefly, roots of intact plants were carefully rinsed in deionized water, incubated in darkness and at room temperature in a continuously aerated assay solution, with the following composition (mM): 0.5 CaSO₄, 10 MES-KOH (pH 5.5), 0.25 Fe³⁺-EDTA, and 0.6 BPDS. After 60 min, the absorbance of the assay solution was determined at 535 nm with a spectrophotometer [73]. The reduced Fe was calculated on the basis of the concentration of Fe²⁺-BPDS₃ complex, using the molar extinction coefficient of 22.1 mM⁻¹cm⁻¹, and was expressed in nmol Fe reduced g_{root FW}⁻¹ h⁻¹.

2.6. Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from approximately 0.1 g of root tissues of tomato plants exposed to different Fe supplies and subjected or not to NPA or AVG treatment, by using Spectrum Plant Total RNA Kit (Sigma-Aldrich Co. LLC), following the operating manual.

Afterward, 1 µg of each total RNA sample was treated with 10 U of DNAse RQ1 and 1 mg of the resulting DNase-treated RNA was utilized to synthesize cDNA by using ImProm-II Reverse Transcription System kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

2.7. Real-Time Reverse Transcription-PCR Analysis

Gene-specific primers were used for the target genes as well as for the housekeeping genes, as previously described by Coppa et al. (2018) [62] and Zuchi et al. (2015) [74]. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) experiments were performed in biological triplicates and the reaction was performed by using the SsoFast EvaGreen Supermix (Bio-Rad), as described in Valentinuzzi et al. (2015) [75].

The amplification efficiency was calculated from raw data using LinRegPCR software [76]. The relative expression ratio value was calculated for treated samples relative to the corresponding untreated sample at the same time-point according to the Pfaffl equation [77]. Standard error values were calculated according to Pfaffl et al. (2002) [78].

2.8. Statistical Analysis

The results are presented as means ± standard error (SE). Statistical analyses were carried out using R software. Statistical significance was tested by one-way ANOVA analysis with Tukey post hoc tests at p < 0.001 and by Student’s t-test.

3. Results

3.1. Effect of Localized Fe Supply on Plant Growth and Chlorophyll Content

Root biomass accumulation, measured in terms of fresh weight, was significantly increased by approx. 40% in the compartment featuring Fe deficiency as compared to that Fe-resupplied (Figure 2A,B). Similarly, plants treated with either NPA or AVG, displayed a higher accumulation of biomass in the Fe-deficient root compartment, independently from the treated side of the root system (i.e., Fe-sufficient or Fe-deficient), as compared to the Fe-sufficient one (Figure 2A,B).

In addition, plants supplemented with NPA showed a slight increase in the allocation of biomass at shoot level (Figure 2C), whilst no significant differences were detected in plants treated with AVG (Figure 2D) as compared to control untreated plants.

Finally, the NPA treatment did not induce any alteration in the concentration of chlorophyll, estimated as SPAD units (Figure 2E); on the other hand, tomato plants supplemented with AVG displayed a slight increase in the chlorophyll concentration (Figure 2F).

3.2. Effect of Localized Fe Resupply on Fe-Starved Plants: ⁵⁶Fe and ⁵⁷Fe Accumulation in Shoots and Roots

In the portion of the root system exposed to Fe shortage, the concentration of natural Fe (⁵⁶Fe) doubled, while that of ⁵⁷Fe was reduced by 93% with respect to the relative Fe-sufficient control portions of the same plant (Figure 3A,B, respectively) at 7 days after ⁵⁷Fe treatment (Figure 1A). On the other hand, ⁵⁷Fe accumulation in the Fe-sufficient (+Fe) root part was 15-fold higher (Figure 3B) compared to that of natural Fe (Figure 3A), and the natural Fe accumulation in the Fe-deficient (-Fe) root part was 2-fold higher (Figure 3A), as compared to that of ⁵⁷Fe (Figure 3B).

In the shoots the concentration of ⁵⁷Fe reached values approximately 32-fold higher than those measured for natural Fe in the same tissue of the same plant (Figure 3A,B, respectively).

3.3. Effect of Localized Fe Resupply on Fe-Starved Plants: Fe³⁺-Chelate Reductase Activity

The in vivo assay of Fe³⁺-chelate reductase enzyme (FCR) on split-root tomato plants showed a significant increase of this activity in roots resupplied with 40 µM Fe³⁺-EDTA as compared to -Fe roots, independently from the treatment applied (Figure 4A,B). In particular, Fe reduction rate was decreased by about 40% in Fe-deficient roots as compared to those Fe-resupplied (+Fe) (Figure 4A,B). Interestingly, when plants were treated with NPA the reductase activity was reduced as compared to control plants (Figure 4A), albeit to a different extent as a function of the compartment supplied with the auxin transport inhibitor. In fact, if NPA was added to the Fe-resupplied compartment, the reductase activity showed a decreasing trend, yet not statistically significant as compared to controls, but, when NPA was applied to the -Fe root compartment, Fe reduction activity was significantly inhibited in both compartments, as compared to the respective controls (Figure 4A). Conversely, when plants were supplemented with AVG, the inhibitor of ethylene synthesis, the resupplied root compartment showed a Fe³⁺ reduction activity higher than the respective untreated control irrespective of the treated root part (i.e., resupplied or not resupplied) (Figure 4B). In particular, when AVG was added to the deficient compartment, the Fe-deficient roots displayed a significantly higher FCR activity as compared to the respective control (Figure 4B); on the other hand, the supply of AVG to the resupplied root portion did not impact the Fe³⁺ reduction activity of the Fe starved root apparatus compared to the control (Figure 4B).

3.4. Effect of Localized Fe Resupply on Fe-Starved Plants: SlFRO1 Gene Expression

Figure 5 shows the expression profile of SlFRO1 (FERRIC REDUCTASE OXIDASE 1) gene, which encodes for Fe³⁺-chelate reductase enzyme. In particular, the expression of the gene followed the same profile reported for enzyme activity in all conditions, except in the NPA-treated plants (Figure 5). In fact, in control conditions, the expression level of SlFRO1 was upregulated in the portion of root system exposed to Fe sufficiency with respect to the -Fe root portion. The same trend was observed also in the other conditions imposed, except for the Fe-sufficient root system supplemented with NPA. Surprisingly, NPA treatment resulted in higher SlFRO1 expression in the Fe-deficient compartment, irrespective of the treated root part (i.e., resupplied or not resupplied) (Figure 5).

4. Discussion

Iron (Fe) is an essential nutrient to plants [15] and, despite its relatively high concentrations in the soil, the bioavailability to plants can be often limited, depending on chemical and physical characteristics of the soils themselves [79,80]. In addition, the bioavailability of nutrients to plants can be also affected by the distribution of mineral elements in the soil, which has been demonstrated to be variable in space and time [1,4,5]. In the specific case of Fe, a few pieces of research have been aimed at investigating the effects of a heterogeneous nutrient supply in dicot plants. The Fe transporter (Iron-Regulated Transporter 1, IRT1) and the Fe-chelate reductase (FCR) enzyme have been demonstrated to be modulated by both local and systemic regulation pathways in Arabidopsis [19]. In addition, the use of the split-root technique in cucumber plants allowed to highlight that both the transcripts coding for CsFRO1 and CsIRT1 as well as the FCR activity were upregulated in the resupplied portion of the root system, whereas they were repressed in the non-resupplied one [58,81]. These observations suggested that Fe acts as local messenger for the upregulation of the uptake system [82]; nonetheless, the downregulation of Fe deficiency response observed in the non-resupplied portion of the root is thought to be mediated by a systemic signal, which could be represented by either the increase in the Fe translocation from the shoots [81] or by other mobile chemical effectors, as for instance phytohormones [29,31,83,84], or by the integration/interplay of both. As already demonstrated by Valentinuzzi et al. (2020) [81], upon resupply, the translocation mechanisms are activated by the availability of the micronutrient, thus causing the allocation of Fe to the starved organs (i.e., leaves and non-resupplied roots). In particular, it was established that, upon resupply, Fe was distributed in the short period (i.e., 12 h) to both starved roots and shoots to overcome the Fe deficiency threshold; however, in the longer period (i.e., 24–48 h), Fe was then preferentially delivered to leaves [81]. Indeed, the resupply data presented in this study (Figure 1A and Figure 3) confirm previous observations, albeit disclosing interesting pieces of information concerning Fe dynamics in split-root systems. In fact, the Fe concentrations detected in the shoots and in the non-resupplied roots of tomato plants after 7 days of treatment with ⁵⁷Fe (Figure 3) were comparable to those previously obtained in cucumber plants after 48 h resupply [81]. This observation suggests that Fe redistribution after the resupply might reach a “steady state” in a short time period (e.g., 24 – 48 h), that is also maintained in the long term (e.g., 7 days). Consistently, this evidence also highlights the importance of shedding light on the signaling events taking place within the short time period after the resupply in order to deepen our understanding of Fe dynamics in plants.

The role of auxin in the response to Fe deficiency has been earlier addressed for its contribution in altering the root morphology in response to the nutritional stress [52]. However, contrarily to previous observations [85], the presence of auxin transport inhibitor NPA did not affect the growth of the root system in the split-root tomato plants, as well as other biometric parameters, independently from the treated root portion (resupplied or not resupplied) (Figure 2A,C,E). The lack of NPA effects on biometric parameters of tomato plants could be ascribable to the short time period of the treatments. More recently, auxin has also been shown to be involved in regulating the molecular response towards Fe starvation, through the transcriptional modulation of Fer-like Iron Deficiency-Induced Transcription Factor (FIT) and FRO2 in Arabidopsis [63]. Indeed, the FCR enzyme activity was influenced by NPA (Figure 4A); in particular, when the inhibitor was added to the non-resupplied portion of the root system, a significant reduction in the FCR activity was observed in both the resupplied and non-resupplied tracts of the root system. Interestingly, this decrease was also mirrored by lower SlFRO1 mRNA levels as compared to control plants, at least in the resupplied root portion (Figure 5); surprisingly, an increase in SlFRO1 expression was displayed locally (non-resupplied roots). In spite of the slight and not significant reduction of FCR activity observed in the tomato plants treated with NPA in the resupplied root portion (Figure 4A), SlFRO1 expression was strongly repressed locally (i.e., in the resupplied root tract) and upregulated in the non-resupplied compartment (Figure 5). The latter observation further confirms the implication of auxin in influencing the regulations of FRO1 [86], yet disclosing a possible different role depending on the nutritional conditions of the root system portion. Furthermore, the role of auxin could be affected by the presence of other hormones and/or signaling substances involved in the participation of the intricate Fe deficiency signaling [33].

Similarly to auxin, ethylene, which is produced in roots experiencing Fe deficiency, has been demonstrated to play a role in the activation of Fe deficiency response [50,87,88,89]. In fact, the supplementation of ethylene synthesis inhibitors (e.g., cobalt, AVG) can prevent most of the physiological responses to Fe deficiency in Strategy I plants, as for instance the FCR activity [30,50,56,87,90]. In our experimental system, the treatment with ethylene inhibitor AVG did not induce any alteration in the biomass accumulation in tomato plants (Figure 2B,D,F). Nonetheless, the presence of the inhibitor, independently from the root tract treated, caused an unexpected increase in the FCR activity in the resupplied roots as compared to control (Figure 4B) [30,50,56,87,90]. However, the higher Fe³⁺ reduction rates observed in the resupplied root tracts as compared to control were not sustained by an enhanced SlFRO1 expression (Figure 5), as also previously observed in literature [30,50,56,87,90]. Interestingly, the effects of AVG on SlFRO1 expression in the non-resupplied root tract was dependent on the treatment; in fact, when the inhibitor was provided in the Fe resupplied root portion, the gene was induced in the Fe starved root portion (Figure 5). On the other hand, if AVG was added to the non-resupplied root zone, SlFRO1 was repressed both locally and in the other half of the root system (i.e., the resupplied one).

In conclusion, the data presented here disclose a very complex activity of both auxin and ethylene in plants provided with a heterogeneous Fe supply. Indeed, both a systemic and local action has been revealed for the two phytohormones. Fully active hormone signaling pathways seemed to be required for the expression of SlFRO1 gene in the resupplied root tracts; nonetheless, the inhibition of SlFRO1 expression in the Fe-sufficient root compartment induced its upregulation in the starved compartment, suggesting that both auxin and ethylene, and/or other secondary messengers, might be involved in this modulation. However, when the signaling pathways were inhibited in the non-resupplied root compartment, auxin showed just a systemic influence on SlFRO1 expression, whereas the ethylene signaling was effective at both local and systemic level. In addition, the investigation of the Fe concentration within tissues highlighted that, even when provided with heterogeneous Fe distribution, plants can take up the micronutrient from the nutrient hot-spots and preferentially translocate it towards shoots, thus rapidly reaching and subsequently maintaining a “steady state”. Indeed, the data presented here indicate that the physiological adaptation to such heterogeneous Fe supply can be mediated by the integration of the complex signaling pathways elicited by both auxin and ethylene activities.

The study of the effect of hormones on plants has aroused great interest among scientists for their role in stimulating plant growth, development, and fruit-set. For this reason, they are widely used by farmers to improve both production and quality of vegetables, including tomatoes [91]. Therefore, from a practical and applicative point of view, the insights in this work could contribute to a more detailed understanding of how hormone modulation can influence the physiology of mineral nutrition in field cultivated plants, having, as a consequence, significant impacts on both the yield and the quality of agricultural products.