1. Introduction
Iron (Fe) is the fourth-most-abundant nutrient on earth, and one of the most important nutrients for biological systems [
1,
2]. It is involved in the energy generation and utilization of plants and plays an important role in many redox reactions, and the absorption, distribution, and storage of Fe by plants are strictly regulated, so the transfer and absorption of Fe in leaves is the focus of the metabolism of the photosynthetic mechanisms [
3]. Fe is primarily present in two forms: ferrous (Fe
2+) and ferric (Fe
3+). Fe
2+ is more soluble and more easily oxidized, forming sediment with inorganic anions [
4]. The solubility of Fe
3+ decreases with increasing pH, mainly due to the lower solubility of hydroxide polymers, which in most cases cannot satisfy the needs of plants [
5]. Since the concentration of free OH
– ions exceeds the Fe
3+-OH formation threshold at neutral pH, maintaining a reduced pH environment and, thus, providing accessible Fe
2+ for metabolic processes is critical [
6]. Thus, the variable pH circumstances in a biological system necessitate multiple strategies for Fe–plant-species coordination [
3]. Plants have devised two strategies to solubilize Fe. The reduction-based strategy, or so-called strategy I, is observed in most flowering plants. Graminaceous species display the chelation-based strategy, or strategy II [
7]. The plants of strategy I take up Fe in roughly three steps: the roots release protons to increase the solubility of Fe
3+, the Fe
3+ is converted to Fe
2+ by ferric chelate reductase (FCR), and then the Fe
2+ is absorbed by the roots through the iron-regulated transporters (
IRT1) [
8]. The plant Fe-absorption system of strategy II synthesizes and secretes mugineic acid (MA) by S-adenosylmethionine (SAM) through an enzymatic reaction [
9,
10], and the enzymes involved in this synthesis mainly include nicotinamide synthase (NAS), nicotianamine aminotransferase (NAAT), and deoxymugineic acid synthase (DMAS) [
11]. The Fe
3+ in soils or media forms chelates with MA and is absorbed into cells through yellow stripe-like (YSL) transport.
In the case of Fe deficiency, the photosynthesis of plants is affected, as well as the absorption and accumulation of nutrients, and multiple signaling molecules, such as H
2S, ethylene, and nitric oxide (NO), are involved to modulate the deficiency [
12,
13,
14]. As a biologically active free radical, NO participates in and regulates the physiological functions of plants. NO is closely related to the process of Fe absorption, transport, and reoxidation. It can stimulate the Fe chelate reductase, significantly up-regulate the gene expression of ferric reduction oxidase 1 (
FRO1) of Fe
3+ and transporter
IRT1 of Fe
2+, thereby increasing Fe absorption and homeostasis [
15]. Fe is reduced to the ferrous form at the root surface and transferred to the leaf in a combined form via the xylem. This suggests that the encoded membrane transporter is also active in the aerial parts of the plant, even though MAs are predominantly found in the roots [
4]. The acquisition in the plant of Fe and other micronutrients starts in the apoplast of the epidermal cells in roots [
16]. The Fe is released into the xylem vessels on behalf of a transfer from symplasm to the apoplast [
17,
18]. Transporters of ferric–citrate, iron–nicotianamine, or other Fe complexes must moderate Fe absorption from xylem vessels, and photoreduction of the xylem carrying Fe carboxylate appears to play a key role in decreased Fe levels in the aerial parts [
19]. The majority of Fe that reaches the leaves are from the Fe absorbed by the roots. The sink-source Fe distribution through the phloem is thought to provide the basis for Fe translocation towards the youngest leaves [
20,
21].
When plants are Fe-deficient, photosynthesis is affected, and chlorophyll cannot be produced normally; typical Fe-deficiency symptoms are the yellowing of young leaves [
1]. In the tetrapyrrole biosynthesis pathway, Fe affects the development of coproporphyrin as well as the formation of chlorophyll [
22,
23,
24]. Moreover, Liu et al. [
25] also found that the expression of deoxyhypusine synthase (DHS) protein in
P. hybrida was related to photosynthesis, and the chlorophyll level was significantly reduced in DHS-silenced leaves of
P. hybrida, showing a chlorotic leaf phenotype. Moreover, Fe deficiency in plants also affects the production and metabolism of carotenoids and anthocyanins. They are synthesized in chloroplasts and play a vital role in the integrity of photosynthetic organs. There is also an inseparable relationship between the anthocyanins and the pH of the substrate [
26]. The anthocyanin-production pathway is regulated by transcription factors, such as v-myb avian myeloblastosis viral oncogene homolog (MYB), basic helix-loop-helix (bHLH), etc. [
27,
28,
29,
30]. Most of the main biochemical reactions that participate in Fe metabolism are completed in the chloroplast, such that the chloroplast is the Fe pool in the plant cell. Fe deficiency will inhibit the conduction of photosynthetic electron chains in the chloroplast and cause optical damage [
31].
More than 70% of the cations and anions absorbed by plants contain ammonium (NH
4+) and nitrate (NO
3−) [
32], and the absorption and accumulation of these two nitrogen (N) sources affect the pH of the plant rhizosphere and apoplast [
33], thus influencing the absorption and utilization of Fe. Excessive accumulation of NH
4+ in plant cells results in toxicity and decreased photosynthesis, and symptoms such as hindered growth, chlorosis, etc., appear [
34]. However, NO
3− accumulation in the rhizosphere increases the pH, reduces the Fe reductase activity, and inhibits Fe transport from root to shoot. Plants possess a regulatory system used for coordinating the uptake and distribution of these nutrients. In the cultivation process of
A. thaliana, the nitrate and/or glucose supplemented to the medium has been shown to alter the levels of carbon (C) and N metabolic genes [
35,
36]. Therefore, in this study, the contents of NH
4+ and NO
3−, and the expression of transport genes were analyzed to explore the indirect effects of Fe and pH on the absorption of NH
4+ and NO
3− in vitro. During the provision of different contents of NH
4+ and NO
3−, the uptake and transport of Fe in plants were affected. However, the effects on plant uptake and transport of nutrients in vitro were investigated when the same contents of NH
4+ and NO
3− were provided, along with different Fe sources and pHs.
The bedding plant
P. hybrida belongs to strategy I, has a short growth cycle, and is extensively used as a model plant of developmental biology [
37].
P. hybrida is an Fe-inefficient plant that is sensitive to neutral and high pH [
38] and responds by reducing the Fe uptake, which generates chlorosis and growth retardation [
39]. Moreover, petunia is also very sensitive to the surrounding NH
4+ and NO
3− [
40,
41]. In some previous studies, the effects of the Fe source and pH on the growth of shrub ornamental plants
Sorbus commixta [
42] and
Hydrangea macrophylla [
43], as well as the regulation of Fe-handling gene expressions, were studied. Moreover, in previous studies, more attention was paid to the effects of the NH
4+ and NO
3− supply on the Fe absorption of plants, while studies seldom discussed the effects of different Fe sources and pH on plant ammonium and nitrate contents. Therefore, in this study, by providing different Fe sources and pH levels to
P. hybrida, we investigate the effects on the growth characteristics, pigment contents, NH
4+ and NO
3− contents, and their gene expressions.
3. Discussion
Higher plants mainly uptake Fe by the root system. On account of a strong demand for Fe, the photosynthetic system will be established and manipulated in the chloroplast of the photosynthetically active tissue, and Fe is transported from the roots to leaves. Petunia is an Fe-inefficient plant that is sensitive to an environment of neutral or high pH, which would result in reduced Fe uptake, followed by appearance of chlorosis and growth inhibition [
45,
46]. This was also confirmed in this study (
Figure 1), as leaves of petunia showed obvious chlorosis at high pHs. The Fe homeostasis in leaves depends on the binding of Fe to cofactors of oxidoreductases, and the developmental system of plants also determines the direction of the Fe distribution in leaf cells [
3]. In this study, the effects of the Fe source and different medium pH on the plant growth and development of
P. hybrida were obvious. According to the growth parameters in
P. hybrida (
Table 1), the plants with the highest number of leaves were observed at pH 4.70 regardless of the Fe source, including the control group. The pH affects the substance solubility and nutrient availability for plants. It is also confirmed in this study (
Table 6) that the number of leaves has a significant positive correlation with the contents of Mn, Ca, K, and P in leaves. At the same time, it was also found that there was a significant regularity between the pH of the medium and the number of leaves and the content of these elements (
Table 1 and
Table 4). Among the iron sources, high pH corresponds to a lesser number of leaves and the content of these nutrient. H
+ as a cation will exchange positions while competing with other cations. The metal availability is often inhibited, and micronutrient deficiencies are generated for plants in alkaline soils [
47]. Moreover, the solubility of Fe under different pH levels also changes. In neutral pH media, Fe exists in the form of ferric Fe and most of the Fe
3+-OH complexes are cross-linked into insoluble ferrihydrite polymers, therefore, the Fe ion content in the medium is extremely low [
48]. The solubility of FeSO
4 is higher at lower pH (below 5.5), and the solubility of FeSO
4 decreases with the increase in the pH [
49]. Fe-EDTA, a chelated Fe, is unstable at high pH; it is relatively stable at a pH lower than 6.0, and highly unstable when at a pH higher than 6.5. That explains why in
Table 1, when the Fe source was Fe-EDTA, the leaf length and root length of
P. hybrida were the greatest at pH 5.70.
In strategy I plants, reduction of Fe chelates is carried out by FCR, and plants are more likely to reduce Fe with weak Fe
3+-chelates. In some Fe-sensitive species (such as
Vaccinium spp. and
Annona glabra), low FCR activity was found in acidic soils with high organic matter and Fe
2+, where these conditions are more favorable for Fe uptake [
50]. Following the FCR-mediated reduction, Fe
2+ is transported through the plasma membrane into the roots [
51]. Strategy I plants upregulate the expression of FCR enzymes when the Fe content of the medium is limited, while increasing the pH level will enhance the FCR activity [
52,
53], which was similar to the findings in
Figure 3. The FCR activity was the highest in control-6.7 (pH is greater than 6.5) among the three pH levels in the control, because the Fe level in the medium affects the root response mechanisms to improve the Fe uptake [
54]. The excess Fe
2+ produced by FCR is re-oxidized by the electron acceptor, but the chelating agent can be re-oxidized by the catalysis of Fe
3+-chelate [
55]. Furthermore, the Fe availability affects the natural distribution of species and limits the growth of important commercial crops. To prevent the potential high Fe toxicity to plants [
56], plants have evolved a series of protective mechanisms that bind Fe and proteins. Plants will reduce Fe uptake by reducing the FCR activity when the Fe content in the medium is excessive; for example, in
Spinacia oleracea and
Brassica oleracea, the FCR activity of roots decrease with increasing Fe content [
57]. The Fe content in the Fe-EDTA was high on account of Fe-EDTA reducing Fe to Fe
2+ by the reaction of metal charge transfer (
Table 4) [
58,
59]. Therefore, the Fe-EDTA group showed a lower overall FCR activity.
Among the extensive range of metal transporters in plants, NRAMP (natural resistant-associated macrophage protein), YSL (yellow stripe-like), and ZIP (zrt- and irt-related protein) families are thought to participate in the Fe transport [
60]. As a member of the ZIP family,
IRT1 is capable of compensating for some defects in the Fe uptake, and IRT2 is also a homologue of the ZIP family [
61].
IRT1 and
IRT2 uptake ferrous Fe from the medium in Fe deficiency, and its gene expression is induced in the roots as a part of the Fe-deficiency response [
62,
63]. In Fe-deficient
A. thaliana, Fe and other metals are absorbed by
AtIRT1 from the medium to regulate Fe homeostasis, and
AtIRT1 is highly expressed in the epidermal region of the roots [
64] (in strategy I plants, the expression of proton pumping, Fe chelate reductase, and
IRT1 are all significantly increased when Fe is deficient) [
65]. In the EDTA-5.7 treatment in this study, the expression level of
PhIRT1 in
P. hybrida leaves was 6947 times higher than that in the control (
Figure 6A), which may be because when the ligand inside plants is binding to metal cations, the high mobility relative to the lower affinity of the chelated Fe is at a negative location of cell membranes and vessels [
66]. It was also found that in
Zea mays, chelating agents such as EDTA or DTPA will increase the Fe transport in leaves. Brian et al. [
67] discovered that the expression of
FRO1 was low in the Fe-rich leaves of
Pisum sativum, while the expression of
FRO1 was increased in the Fe-deficient leaves [
68]. The highest
PhFRO1 expression and Fe content were observed in EDTA-5.7 in this study (
Figure 6B); before uptake of Fe
3+ by leaf cells,
FRO1 participated in the process of composite reduction of Fe
3+ to Fe
2+ [
67,
69].
FRO8, which is also a chelate Fe reductase, exists in the mitochondria of cells, mainly acts on Fe reduction during shoot senescence [
70], and also participates in the reduction of Fe
3+ to Fe
2+. Simultaneously, the highest expression of
PhFRO8 was observed in EDTA-5.7 (
Figure 6C).
The plant pigment contents in leaves are closely related to the physiological function of leaves, which can provide a valuable insight into the physiological functions of leaves. According to the contents of total chlorophyll (chlorophyll a and b), carotenoids, and anthocyanins in
Figure 2, the contents of these photosynthetic pigments and secondary metabolites were influenced by the Fe source and medium pH. However, the effects of the medium pH were stronger than that of the Fe source. As shown In
Figure 1 of this study, when the pH was 6.70, the leaves were chlorotic. The same trend was also observed in the pigment contents in leaves. This is similar to the results of Smith et al. [
38,
39], where when the pH of the substrate was increased from 5.0 to 6.0, the contents of the total chlorophyll and carotenoids in leaves of
P. hybrida ‘Priscilla’ decreased, chlorosis appeared after 2 weeks, and, when the pH of the medium was raised to 7.0, the contents of total chlorophyll and carotenoid decreased significantly. Chlorosis of young leaves in general is the first visual phenomenon of Fe deficiency. It is associated not only with the damage of chlorophyll but also with changes in the expression and assembly of other components of the photosynthetic organ [
71]. In photosynthetic cells, approximately 80% of the Fe is a key determinant in the formation of prosthetic groups in enzymes [
72]; the Fe content plays a role in a variety of vital plant activities such as chlorophyll production, electron transport between PSI and PSII, and maintenance of the structure and function of the chloroplast [
73].
The function of chloroplasts depends on the plastid and nuclear genomes of proteins encoded, and a normal development of chloroplasts is associated with the leaf color [
74,
75]. The DHS proteins localized in the cytoplasm and nucleus affect the chlorophyll levels in leaves, and in
Solanum lycopersicum and
Nicotiana tabacum L., the formation of deoxyhypusine residues is catalyzed by DHS, which plays a role in chloroplast development [
76]. Liu et al. [
25] observed that in
DHS-silenced leaves in petunia, the chloroplast development had abnormal and reduced chlorophyll levels and activity of photosystem II. However, in our study, the highest expression of the
DHS gene appeared in EDTA-5.7, where the chlorophyll content and chlorosis state were about the median among all the treatments; it is, therefore, speculated that the DHS expression is more related to the pH. Fe reacts to the porphyrin structure of chlorophyll, the main component of the chloroplast. A famous function of the cytochrome is electron transfer, and cytochrome oxidase is involved in the last step of the respiratory chain [
77]. The ratio of condensation for glycine and succinyl-CoA synthesis of ALA (delta-aminolevulinic acid) is reduced when plants are Fe-deficient. At that time, the photosynthetic system remains intact, but the number of photosynthetic units decrease [
78]. Nenoval et al. [
79] found that excess Fe can increase the pigment contents in pea leaves by at least 28%. The relationship between the chlorophyll content and spectral index is also affected by other pigments [
26]. The anthocyanin content tends to be higher in young leaves with low photosynthetic rates [
80], and metal ions and pH can alter the anthocyanin color in vitro but only play a minor role in vivo. The biosynthesis of anthocyanins is mainly regulated by several transcription factors in the R2R3-MYB family, of which
MYB113 mainly regulates the anthocyanin expression in mature leaves [
81]. Several research data indicated that anthocyanin synthesis is enhanced when the
MYB113 gene is overexpressed in
A. thaliana [
30]. It was confirmed in our study that the control-4.7 treatment with the highest expression of
PhMYB113 (
Figure 6F) also resulted in the greatest anthocyanin content (
Figure 2C), indicating that the
PhMYB113 expression of the control group corresponded to the anthocyanin level. However, in treatment groups with FeSO
4 and EDTA, the expression of
PhMYB113 was the highest at pH 5.70. Since nutrients affect the synthesis of anthocyanins and also activate or inhibit the expression of transcription factors [
82], it is speculated that the Fe source and pH affect the expression of
PhMYB113. In vegetative tissues, anthocyanins are considered important photosynthetic antioxidants [
83,
84]. When plants are deficient in anthocyanins, photoprotection is achieved through an alternative mechanism [
85]. Therefore, the change in the anthocyanin content often also represents the diversity of antioxidants [
86]. In the antioxidant processes of plants, H
2O
2 is oxidized by SOD and then scavenged by GPX, POD, and CAT. Under the influence of Fe and ROS, the activities of GPX, POD, and POD change accordingly [
87]. As shown in
Figure 2, the pH affects Fe absorption, resulting in reduced anthocyanin content with Fe deficiency.
Several steps of biosynthesis are dependent on Fe. Fe-deficiency chlorosis severely affects crop quality and production. Meanwhile, chlorosis caused by Fe deficiency in a high pH environment is generally ameliorated by other approaches, such as acidification of the medium or the use of amino-based fertilizers, to lower the pH to achieve higher solubility of microelements [
45,
88]. At the same time, the results of other research indicate that the correlation between the Fe concentration and chlorophyll content is not always the same, as in the case of chlorosis caused by Fe deficiency, and the Fe concentration in chlorotic leaves may be higher than that in non-chlorotic leaves. When the pH is too high, Fe can be fixed in the free space of leaves [
89]. The Fe source and pH not only affect the plant pigment content but also the macronutrient and micronutrient contents [
90]. Floral crops cultivated under soilless conditions are commonly cultivated with a medium pH range, usually controlled between 5.6 and 6.2. In the leaves of plants, the availability of nutrients correlates with the water solubility of the applied compound [
66]. The solubility of nutrients is controlled by the pH. When the pH increases, the solubility of the microelements in the medium decreases, and oxyphilous plants appear as a nutrient-deficiency symptom. Meanwhile, when Fe-efficient plants such as
Pelargonium hortorum and
Tagetes erecta L. are grown in a low-pH medium with high solubility of Fe and Mn, elemental poisoning occurs, thereby generating necrosis in plants [
45]. The high pH of the medium also restricts Mn uptake, and the symptoms of Mn deficiency are similar to those of Fe deficiency [
91]. As shown in
Table 4, in the control and FeSO
4 groups, low Mn content corresponded to a high–medium pH. According to the results of Abadia et al. [
92], when the leaves of
Prunus persica L. appear to be chlorotic, the content of K ions increases, while the content of Ca ions in the leaves decreases, thereby the value of K/Ca is increased. However, as shown in
Figure 1 and
Table 4, chlorosis of leaves was obvious with the increase in the pH, and the content of K and Ca ions also decreased, especially in the control and FeSO
4 groups. It has been reported that the reduction of chlorophyll is not always due to Fe deficiency but may also be related to the dilution effect of leaves, and the appearance of chlorosis is not necessarily directly related to the nutrient contents.
In addition, N is also an important mineral nutrient for crops, and an important factor for plant growth and development [
93]. Some important organic compounds in plants are inseparable from N, such as proteins, chlorophyll, alkaloids, etc. [
41]. N is mainly dissolved in the form of NH
4+ and NO
3−, then absorbed by the roots of plants [
94]. A large proportion of NO
3− and a fraction of NH
4+ are directly transferred to the leaves, and the remainder is assimilated [
94,
95]. The absorption of NO
3− and NH
4+ significantly affected the pH of the rhizosphere and apoplast, thereby affecting the absorption and utilization of Fe by plants [
96]. As shown in
Figure 5 and
Table 6, the nutrient contents interact with each other in
P. hybrida leaves. In the control group under Fe deficiency, lower pH corresponded to lower NO
3− contents, because the increase in pH increased the NO
3− content, while reducing the activity of Fe
3+ reductase, thereby inhibiting the transport of Fe from roots to shoots [
97,
98]. In the treatment with FeSO
4 or chelated iron EDTA, due to supplementation of Fe
2+ or Fe
3+, the interaction effect and absorption by leaves of nutrients were changed. The N uptake of plants is mainly determined by specific genes, such as the nitrate transporter
NRTs and ammonium transporter
AMTs [
99]. The regulation of the
NRT and
AMT expressions are affected by N metabolites, light, pH, sucrose, etc. [
100,
101]. Among them, NRT2.5 is a high-affinity nitrate transporter that is localized in the plasma membrane and mainly expressed in the epidermis of root hairs and leaves [
102]. As shown in
Figure 6D, in the control group, the expression level of
PhNRT2.5 was lower than that in the FeSO
4 and EDTA groups, and the highest expression level was observed in EDTA-5.7. In addition, the overexpression of
AMT1 will increase the absorption of ammonium by plants, but, with sufficient ammonium content,
AMT1 will reduce the biomass of the aboveground parts in order to reduce ammonium toxicity [
94]. As shown in
Figure 6E, the expression of
PhAMT1;1 in the control group was the lowest and was affected by the pH, with high pH corresponding to high expression. The highest expression of
PhAMT1;1 was observed in the EDTA group, and the expression decreased with increasing pH. Therefore, we judged that under the synergistic effects of Fe and pH, the uptake and contents of NO
3− and NH
4+ were affected in
P. hybrida leaves.