Getting a sense for signals: regulation of the plant iron deficiency response

Abstract

Understanding the Fe deficiency response in plants is necessary for improving both plant health and the human diet, which relies on Fe from plant sources. In this review we focus on the regulation of the two major strategies for iron acquisition in plants, exemplified by the model plants Arabidopsis and rice. Critical to our knowledge of Fe homeostasis in plants is determining how Fe is sensed and how this signal is transmitted and integrated into a response. We will explore the evidence for an Fe sensor in plants and summarize the recent findings on hormones and signaling molecules which contribute to the Fe deficiency response.

1. Introduction

Fe is essential for both plant growth and crop yields and most importantly, humans rely on dietary Fe from plant sources. According to the World Health Organization, the current most common nutritional disorder in the world is Fe deficiency, with over 30% of the world's population affected [http://www.who.int/nutrition/topics/ida/en/index.html]. Due to the limited solubility of Fe in most neutral or basic soils, there is not a readily accessible supply of Fe in the rhizosphere and plants are often limited in Fe content. Thus, increasing the ability of plants to acquire and store Fe could have significant effects on plant and human nutrition. With this goal in mind, it is important to uncover the mechanisms of how plants sense and respond to Fe availability.

When faced with Fe limitation, plants employ a set of responses to boost Fe mobilization and uptake from soil so they can ensure there is enough Fe for critical cellular processes [1]. Fe is an essential cofactor in metabolic processes such as the respiratory electron transport chain. Additionally, as photosynthetic organisms, plants require Fe for chlorophyll biosynthesis and for the reactions of photosynthesis. There are two main strategies plants use for Fe acquisition. First, Strategy I, based on reduction of Fe, is used by non-grasses such as Arabidopsis. Second, grasses (also known as graminaceous plants) such as rice use Strategy II, a chelation based strategy. We will briefly outline the basic components of each of these strategies and then discuss in detail the regulation of each of these strategies. For a more comprehensive overview of the two strategies as well as discussion of how our knowledge about these strategies has led to transgenic crops with enhanced tolerance to iron deficiency or with increased iron content, the reader is referred to Kobayashi and Nishizawa [2]. This review will also explore the possibilities for how Fe is sensed and how different signals are integrated into the response, with particular attention to the recent advances in the field.

2. The reduction strategy

Upon Fe limitation, plants which use the reduction strategy release protons via root plasma membrane H⁺-ATPases belonging to the AHA family [3, 4]. As exemplified by Arabidopsis, several AHAs are induced in Fe-deficient roots, but analysis of loss of function mutants suggests AHA2 is the chief player [5]. This acidification by proton release serves to increase the solubility of Fe in the soil. One unit drop in pH increases the solubility of Fe by 1,000 fold [6]. Following acidification, Fe³⁺ is reduced to Fe²⁺ by a membrane-bound ferric-chelate reductase enzyme, i.e. by AtFRO2 in Arabdiopsis or PsFRO1 in pea [7, 8]. Reduction seems to be a rate-limiting step in Fe uptake because transgenic overexpression of ferric chelate reductases in Arabidopsis, rice, tobacco, and soybeans increases tolerance to low iron [9-12]. The reduced form of Fe is transported into the root by the plasma-membrane divalent cation transporter IRT1 [13, 14], the founding member of the ZIP family [15]. IRT1 is an essential gene because irt1 mutants are severely chlorotic and seedling-lethal unless supplied with large amounts of exogenous Fe [16-18]. Expression of IRT1 and FRO2 indicates that Fe uptake occurs predominantly in epidermal layers [16, 19].

Besides these physiological mechanisms, plants respond to Fe deficiency through morphological changes that result in increased root surface area for the reduction and uptake of Fe. Examples include increased formation and branching of root hairs, root-tip swelling, and enhanced lateral root formation [20, 21].

3. The chelation strategy

Grasses release phytosiderophores (PSs), such as mugineic acids (MAs), which bind Fe³⁺ with high affinity, in order to acquire Fe from the rhizosphere in Fe-limited conditions [22]. Phytosiderophores are synthesized from nicotianamine (NA), a non-proteinogenic amino acid formed by condensation of three molecules of S-adenosyl methionine. Although all plants can synthesize NA, which serves as a transition metal chelator, only the grasses go on to convert NA to PS. The chelated complexes of Fe(III)-PS are subsequently transported into the roots through Yellow Stripe (YS)/Yellow Stripe-like (YSL) family transporters, named for YS1 of maize [23, 24]. For example, OsYSL15 is the major transporter responsible for Fe(III)-PS uptake in rice [25, 26]. Other members of the YSL family transport metal-NA complexes in both grasses and non-grasses.

Although the biosynthetic pathway and the uptake transporters have been well studied [2], the mechanism by which PS are released remained unknown. The missing piece was recently identified: two transporters of the major facilitator superfamily (MFS), TOM1 and HvTOM1 from rice and barley respectively, were shown to be involved in the efflux of the PS deoxymugineic acid [27]. Xenopus oocytes expressing either transporter were able to release ¹⁴C-labeled deoxymugineic acid but not ¹⁴C-labeled NA, suggesting TOM1 and HvTOM1 are PS efflux transporters.

In the same study, two other rice MFS members, ENA1 and ENA2, were identified as NA transporters by their ability to transport ¹⁴C-labeled NA, but not ¹⁴C-labeled deoxymugineic acid [27]. ENA1 is similar to AtZIF1, which localizes to the vacuolar membrane and was shown to be involved in Zn detoxification [28]. Although originally thought to be a Zn transporter given its localization and the zinc sensitive phenotype of an atzif1 loss of function mutant, its similarity to ENA1 suggested that AtZIF1 might be a NA transporter. Recently, overexpression of ZIF1 has been shown to enhance NA accumulation in vacuoles [29]. Additionally, heterologous expression of ZIF1 increases NA content in yeast cells expressing nicotianamine synthase, but does not complement a Zn-hypersensitive mutant that lacks vacuolar Zn transport activity. Similarly, ENA1 may participate in metal detoxification by transporting NA into the vacuole.

Despite being a Strategy II plant by uptake of Fe(III)-PS, rice possesses a ferrous transporter, OsIRT1, and can take up Fe²⁺ [30, 31]. Evidence in support of the importance of being able to take up Fe²⁺ comes from a study of rice that cannot synthesize PS due to a mutation in the NICOTIANAMINE AMINOTRANSFERASE (NAAT) gene. This mutant can still grow normally when supplied with Fe²⁺ [30]. Furthermore, lines carrying a T-DNA insertion in the YSL15 transporter gene are viable. Strategy II plants, however, do not have an inducible ferric-chelate reductase activity that is classic to the Strategy I response. This is likely the result of an adaptation to waterlogged rice paddies, in which Fe²⁺ is more prevalent than Fe³⁺ due to reduced oxygen levels [31].

4. Regulation of the reduction strategy

There have been a number of transcriptomic and proteomic studies aimed at determining gene expression and protein profile changes upon Fe deficiency. Proteomic studies are beyond the scope of this review, but for a recent discussion of the Fe deficient protein profile in Arabidopsis, refer to Schmidt and Buckhout [32]. Several recent transcriptomics studies have aimed at identifying regulatory networks involved in Fe homeostasis in Arabidopsis [32-35]. Here, we will discuss two main networks that are currently at the forefront of the field: the FIT network and the POPEYE network. For another recent review on regulation of the reduction strategy, refer to Ivanov et al [36]. Both Ivanov et al. [36] and the study by Long et al. [37] use co-expression analysis to show regulatory networks involved in Fe deficiency responses.

4.1. The FIT network

In Arabidopsis, FIT (FER-LIKE IRON-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR), is required for regulation of the Strategy I Fe-deficiency response [38-41]. FIT is the functional ortholog of FER, a bHLH transcription factor essential for the Fe-deficiency response in tomato [38-41]. In addition to IRT1 and FRO2, FIT is induced in the epidermis of the root upon Fe-deficiency and like the irt1 mutant, fit is seedling lethal unless watered with supplemental Fe [38]. FIT controls the Fe uptake machinery at multiple levels: FRO2 is transcriptionally regulated by FIT, while IRT1 is both transcriptionally and posttranscriptionally regulated by FIT [38, 39]. Recent studies have examined how FIT itself is regulated [42-44]. Sivitz et al. [44] demonstrated a dual regulation of FIT by Fe starvation. At the transcriptional level, FIT is induced by Fe starvation and FIT protein subsequently accumulates. The second mode of regulation is post-transcriptional. Using proteasomal and translational inhibitors, they showed that in Fe-limited conditions, FIT is actively destabilized and turned over by 26S proteasomal degradation. The authors suggest that in Fe deficiency, FIT binds to its target promoters and then this “exhausted” FIT is rapidly degraded. “Fresh” FIT is synthesized to allow for subsequent transcriptional cycles and amplification of FIT target gene transcription. This idea, that a limited half life of transcriptional activators may promote continuous gene expression by delivery of “fresh” activator after “fatigued” activator is spent, has previously been described [45]. Alternatively, a more simple explanation is that the decreased stability and increased proteasomal degradation of FIT in Fe-deficient conditions is to ensure that once synthesized, FIT can be rapidly removed from the cell. The function of this would be to prevent FIT from activating gene expression when it is no longer needed (i.e. when Fe supply increases).

Regulated turnover and activity of FIT strengthen the notion that FIT is a major transcriptional regulator of the Fe deficiency response. Evidence suggests FIT is a key regulator in integrating incoming hormonal and other intracellular signals. For example, ethylene signaling effects FIT abundance via two transcription factors in the ethylene signaling pathway [42]. This model for FIT turnover will be discussed in section 7.1.3. Another signaling molecule, NO, has also been implicated in posttranslational regulation of FIT [43]. It is proposed to play a role in stabilization of FIT protein. These findings will be discussed with further detail in section 7.1.5. Overall, control of FIT turnover and activity may be the major way plant roots respond to changing Fe availability in the rhizosphere and meet the nutritional demand for Fe.

Overexpression of FIT does not affect FRO2 and IRT1 expression in the root, suggesting that FIT may act with a binding partner to form a heterodimer [38-40]. qRT-PCR experiments have shown that mRNA for four additional bHLH genes (bHLH38, bHLH39, bHLH100, and bHLH101) are induced by Fe-deficiency [40, 46, 47]. Using bimolecular fluorescence complementation, FIT was shown to interact with both bHLH38 and bHLH39 [47]. In plants constitutively overexpressing both FIT and bHLH38 or bHLH39, FRO2 and IRT1 expression are high and plants exhibit greater Fe accumulation than WT [47]. These data support a role for FIT heterodimer formation with either bHLH38 or bHLH39 for induction of the strategy I response.

In a recent report [47], co-overexpression of FIT and bHLH38 or bHLH39 was shown to increase expression of HMA3 and IRT2 in addition to MPT3 and IREG2/FPN2, which are previously published FIT targets [38]. These genes encode proteins that function in sequestration of heavy metals in vacuoles and vesicles. One result of such overexpression is increased Cd sequestration in roots and therefore less Cd accumulation in the shoots. Additionally, the overexpression lines exhibit increased NICOTIANAMINE SYNTHASE 1 and NICOTIANAMINE SYNTHASE 2 (NAS1 and NAS2) expression, which results in enhanced NA accumulation. The increase in NA causes an increase in transport of Fe from root to shoot. Both the increase in Cd storage in the roots and an increase in shoot Fe content may serve to alleviate Cd toxicity.

4.2. The POPEYE network

High resolution expression profiling has been used by the Benfey laboratory to report how each cell layer in the root responds to Fe deficiency [19]. Although expression of most genes related to Fe uptake are increased in the epidermal tissue and are represented by the FIT regulatory network, another set of transcriptional changes upon Fe deficiency occurs in the vasculature and may represent another transcriptional network. Two genes from this additional regulatory system have been investigated in more detail. These are the recently identified bHLH transcription factor (bHLH047) named POPEYE (PYE) and the putative E3-ubiquitin ligase called BRUTUS (BTS) that are both induced in Fe deficiency with their highest expression in the pericycle of root [37]. However, PYE protein was localized to the nuclei of all cells within Fe-deficient roots, suggesting that after induction by Fe-deficiency within the pericycle, PYE may move throughout the root. pye-1 exhibits chlorosis and root growth inhibition in Fe-deficient conditions, suggesting it is an important regulator of the Fe deficiency response.

ChIP-on-chip analysis reveals that PYE targets key genes implicated in metal homeostasis including NAS4, FRO6, and ZIF1, which encode a nicotianamine synthase, a plasma membrane-localized ferric chelate reductase from the same family as FRO2, and a vacuolar-localized transporter important for Zn tolerance [28, 49, 50]. In pye-1, expression of these genes is significantly increased and prolonged upon Fe deficiency, suggesting that PYE normally acts to repress the activity of these metal homeostasis genes [37].

bHLH proteins, like FIT, often form dimers that interact with downstream targets [47]. Therefore, it is likely that the bHLH protein PYE may act with a binding partner. Yeast-two-hybrid studies suggest that PYE indirectly interacts with BTS through PYE homologs, notably two additional bHLHs: ILR3 and bHLH115 [37].

Analysis of the partial loss-of-function bts-1 mutant, which exhibits increased root length and increased rhizosphere acidification in Fe deficient conditions compared to WT, suggests that BTS is a negative regulator of the Fe deficiency response [37]. This study contains the only reported data on BTS to date and it is clear we need more information to understand how BTS is functioning. The authors suggest that PYE, PYE homologs, and BTS form a regulatory network for maintaining Fe homeostasis in low Fe conditions, but this hypothesis requires in planta confirmation.

5. Regulation of the chelation strategy

5.1. Positive regulation by OsIRO2 and OSIDEF1/2

Using microarray expression profiling in rice, the bHLH protein OsIRO2 was identified as a transcription factor that is upregulated upon Fe deficiency [51]. Unlike FIT/FER, of which there appears to be no counterpart in rice, IRO2 expressed in both roots and shoots in Fe deficient conditions. When OsIRO2 is overexpressed, plants grow better than WT under Fe-deficient conditions while RNAi knocked-down OsIRO2 plants do worse than WT. Correspondingly, the overexpression lines have enhanced expression of genes involved in PS synthesis and transport while the RNAi lines have diminished expression of the same genes. In these plants, OsIRT1 levels remain unchanged, suggesting that OsIRO2 is responsible for regulation of PS-mediated Fe uptake, but not Fe(II) uptake. Additionally, not all genes regulated by OsIRO2 have the characteristic consensus IRO2 binding sequence (CACGTGG), so IRO2 may act by regulating other transcription factors. In another recent study, promoter-GUS lines demonstrated that the expression pattern of OsIRO2 is very similar to downstream genes such as OsNAS1 and OsNAS2 in embryos during seed development and germination and in vascular bundles in roots and leaves during vegetative stages [52]. As previously reported, OsIRO2 overexpression lines exhibited improved tolerance to Fe deficiency. Additionally, seeds accumulated higher levels of Fe compared to WT when plants were grown in alkaline soil. The results of this study suggest that OsIRO2 is synchronously expressed with other genes involved in Fe homeostasis and plays a key role in Fe uptake and Fe transport in both germination and seed development.

OsIDEF1 and 2 (IDE binding Factor), members of the ABI3/VP1 and NAC transcription factor families, respectively, have also been identified as positive regulators of the Fe deficiency response based on their ability to bind to sequences that confer Fe regulation on the barley root-specific IDS2 promoter [53]. These sequences are called iron-deficiency-responsive elements 1 and 2, or IDE1 and IDE2 [53]. Interestingly, sequences homologous to IDE1 are also found in other Fe deficiency inducible promoters, such as OsNAS1, OsNAS2, OsIRT1, AtIRT1, and AtFRO2. OsIDEF1 and OsIDEF2 bind to specific sequences within IDE1 and IDE2: CATGC anCA[A/C]G[T/C][T/C/A] [T/C/A], respectively. OsIDEF1 transcripts are constitutively expressed and levels do not depend on Fe status [54]. Rice plants with RNAi knocked-down OsIDEF1 are susceptible to early-stage Fe deficiency, while rice overexpressing OsIDEF1 are more tolerant to Fe-deficiency [55, 56]. Time course expression analysis revealed that on the onset of Fe starvation, OsIDEF1 positively regulates the induction of several known Fe uptake and utilization genes in rice, such as OsIRO2, OsYSL15, OsYSL2, OsIRT1, OsNAS1, OsNAS2, and OsNAS3 [55]. As Fe deficiency progressed, activation of these genes by OsIDEF1 was limited. As for OsIDEF2, reduction of expression by RNAi results in change in only one gene previously implicated in Fe homeostasis: OsYSL2, which encodes a metal-NA transporter [57]. Unlike OsIDEF1, OsIDEF2 does not appear to change its effects on downstream targets as Fe deficiency progresses [56]. These observations indicate a differential response to early and subsequent Fe deficiency. Taken together, the data suggest OsIDEF1 may be a very upstream regulator in the Fe-deficiency response. Kobayashi et al. [58] suggest the possibility that OsIDEF1 may function to sense the cellular Fe status at the beginning of Fe deficiency. We will report their findings in section 6 of this review.

5.2. Negative regulation by bHLH OsIRO3

Through analysis of transcriptome data from Fe-deficient rice, another bHLH TF has just been identified for its role in Fe homeostasis [59]. This TF, OsIRO3, is greatly induced both in root and shoot in Fe deficiency. Rice plants overexpressing OsIRO3 were hypersensitive to Fe deficiency and accumulated less Fe in shoots than WT. Additionally, genes normally induced in Fe deficiency such as OsNAS1, OsNAS2, OsIRO2, OsIRT1, OsYSL15, and OsNRAMP1 are no longer induced in OsIRO3 overexpressing plants. Together, these results indicate that OsIRO3 is a negative regulator of the Fe deficiency response in rice and acts upstream of OsIRO2.

Interesting, phylogenetic analysis shows that OsIRO3 is the most similar of three rice orthologs to POPEYE in Arabidopsis [59] (Refer to Figure 1). Both PYE and OsIRO3 seem to be involved in negatively regulating gene expression. In pye-1, several Fe homeostasis genes are up-regulated including bHLH39, bHLH101, OPT3, FRD3, NRAMP4 and as mentioned previously, NAS4, FRO3, and ZIF1 [37]. For OsIRO3, only overexpression lines have been analyzed [59]. Knocking down OsIRO3 in rice would allow us to more directly compare the effects of OsIRO3 and POPEYE. It will be interesting to see if these two orthologous genes play similar roles in the Fe deficiency response in rice and Arabidopsis.

Figure 1. Phylogenetic tree of bHLH transcription factors important for the Fe deficiency response in Arabidopsis and rice.

The bHLH transcription factor family is greatly expanded in plants (162 bHLH genes in Arabidopsis and 111 in rice compared to 8 in Chlamydomonas; [107]) and several members are implicated in Fe homeostasis as discussed in this review. Evolutionary history was inferred using Maximum Likelihood analysis conducted in MEGA5 [108]. The bootstrap consensus tree displayed here was inferred from 1000 replicates. Values represent percentage of replicate trees in which the associated proteins are clustered together. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

6. The Fe Sensor

Grafting and split root experiments suggest that there are both local and systemic signals involved in the Fe deficiency response in plants [60-62]. Signals must be then transmitted by a signal transduction cascade that ultimately results in transcriptional regulation of downstream effector genes of the Fe uptake system. Although there are several candidates for a long distance Fe signal in plants, including the metal chelator NA [63], the identity of a local Fe sensor remains unknown.

In other systems, there is evidence for such a master Fe sensor. For example, in many species of bacteria, the Fe-binding ferric uptake regulator (Fur) protein is responsible for Fe²⁺-dependent transcriptional repression of Fe-regulated genes [64]. Because Fur directly binds Fe, it serves as an Fe sensor. The Fe sensor in yeast is unknown, but evidence supports a role for mitochondrial Fe-S cluster formation and cytosolic monothiol glutaredoxins in the sensing mechanism [65-67].

In mammals, Fe regulatory proteins (IRPs) govern cellular Fe homeostasis by regulation of translation and stability of mRNAs involved in Fe homeostasis [68]. In a state of low Fe, IRPs bind to Fe response elements (IREs) in the 5′ or 3′ untranslated regions of mRNA. Binding in the 5′ region causes translational repression, while binding in the 3′ region causes mRNA stabilization. A well-characterized example is the binding of IRP to the 5′ region of ferritin mRNA under low Fe conditions. This results in decreased synthesis of ferritin, which halts sequestration of Fe. In general, in low Fe conditions, IRPs bind to IREs to increase bioavailable Fe by increasing uptake and decreasing Fe utilization and storage. Conversely, when cells have excess Fe, IRPs do not bind to IREs, reducing Fe levels by decreasing uptake and increasing utilization and storage. There are two IRPs: IRP1 and IRP2. Fe governs the activity of each of these IRPs in a different manner. In Fe-sufficient conditions, Fe binding to IRP1 leads to formation of an Fe-S cluster and IRP1 acts as a cytosolic aconitase. This Fe-S cluster inhibits IRP1 from binding to IREs in mRNA. In Fe deficient conditions, IRP1 is free to act on mRNAs. IRP2, other hand, does not form an Fe-S cluster in Fe sufficient conditions but rather is ubiquitinated and degraded by the proteasome. Until the recent work of two independent labs [68, 69], the identity of the E3 ligase responsible for IRP2 degradation was unknown. Each group elucidated the function of FBXL5 in a different manner. Vashisht's group focused on uncovering new roles for mammalian F-box proteins [69]. An F-box protein is a member of an SCF (Skp1, Cullin1, and RBX1)-type E3 ligase and is responsible for tethering a target protein to an E3 ligase complex that tags a protein for ubiquitination and degradation by the proteasome. In this study, the F-box domain of FBXL5 was deleted. This deletion construct was expressed in cells and because it could avoid degradation without an F-box domain, it was used to trap its targets. IRP1 and IRP2 were then identified by mass-spectrometry as FBXL5 binding partners. Saluheeden's group used RNA interference to decrease expression of specific E3 ligase components in cell culture [68]. In cells treated with Fe, IRP2 degradation occurred. However, in cells lacking FBXL5 or any SCF components, IRP2 was spared from degradation.

Both groups identified a conserved hemerythrin domain in FBXL5 [68, 69]. Hemerythrin domains are cation binding motifs known to bind Fe and oxygen at their center [70]. Previously hemerythrin domain-containing proteins were identified in invertebrates and bacteria, but were not known to exist in higher life forms [71]. Both groups demonstrate that binding of Fe to the hemerythrin domain stabilizes FBXL5, while lack of Fe destabilizes FBXL5, resulting in its degradation [68, 69]. This leads to accumulation of IRPs and their downstream translational activity that increases Fe uptake and decrease Fe storage. Although both IRP1 and IRP2 can be targeted by FBXL5, as mentioned previously, IRP1 has the ability to form Fe-S clusters to prevent it from Fe-dependent degradation. This work has identified FBXL5 as the mammalian Fe sensor.

In plants, although it has been long sought, no Fe sensor has been identified. Fe-S cluster formation as in IRP1 has not so far been demonstrated in plants [72]. However, putative Fe-binding domains have been identified in both Arabidopsis and rice [37, 58]. The proteins containing these domains warrant further study as they may act as local sensors that bind Fe to transmit the cellular Fe status for downstream transcriptional regulation. In Arabidopsis, the putative RING-type E3 ligase BTS contains hypothetical hemerythrin domains [37]. Therefore, it is likely there is an ubiquitin-dependent proteolysis system involved in Fe regulation. However, it is clear that BTS is not acting in an identical manner to FBXL5. First, BTS belongs to a different family of E3 ligases and contains 6 hemerythrin domains, rather than the one identified in FBXL5. Probably more importantly, BTS is induced in Fe-deficient conditions [37] whereas FBXL5 mRNA accumulation is independent of Fe status [68, 69]. There are presently no published data on BTS protein accumulation as a function of Fe status, but as mentioned previously, FBXL5 is stabilized by binding of Fe to the hemerythrin domain in Fe-sufficient conditions. In Fe-deficient conditions, the hemerythrin domain and thus FBXL5 are unstable. If BTS protein accumulation corresponds to BTS expression in Fe-deficient conditions, then BTS and FBXL5 accumulation would be opposite for plants and mammals. If this is the case and BTS is only present in Fe-deficient conditions, it is unlikely that Fe binding can stabilize hemerythrin domains and BTS itself as occurs with FBXL5.

The data that bts-1 performs better in Fe-deficient conditions than does WT suggests it may be a negative regulator of the Fe deficiency response [37]. Therefore, its mode of action as an E3 ligase may be the ubiquitin-dependent degradation of proteins positively regulating the Fe-deficiency response. However, BTS' activity as an E3 ligase and its hemerythrin domains remain largely uncharacterized. Further analysis of BTS may provide important insight into how Fe status is perceived in Arabidopsis.

In rice, OsIDEF1 may be a candidate Fe sensor. As mentioned previously, OsIDEF1 mRNA levels are not regulated by Fe status and OsIDEF1 positively regulates a set of Fe uptake and utilization genes early in the Fe deficiency response [55, 56]. In rice, histidine-asparagine (His-Asn) repeats and proline-rich regions were identified in both OsIDEF1 and its ortholog in barley, HvIDEF1 [58]. Affinity chromatography demonstrated that these domains in OsIDEF1 bind to Fe²⁺ and other divalent metals such as Ni²⁺, Zn²⁺, and Cu²⁺ with no particular specificity. Some impairment of transcriptional regulation in the early Fe-deficiency response occurred as a result of deleting these regions, suggesting that OsIDEF1 binds metals and senses changes in Fe availability. The authors propose a model in which OsIDEF1 binding of metals by His-Asn and proline-rich domains exists in an equilibrium between binding Fe, binding other metals, and in a metal-free form [58]. They suggest OsIDEF1 may detect the ratio of Fe to other metals to sense the progression of Fe deficiency. Basically, under Fe-sufficient conditions, the ratio of Fe to other metals would be high. Upon the onset of Fe deficiency, the ratio would decrease and OsIDEF1 target gene regulation may change. Subsequently, as Fe deficiency progresses, the ratio decreases further. Again, such a change may alter the influence OsIDEF1 has on its target promoters. The question remains how OsIDEF1 transmits a metal-binding signal to its downstream pathways. Deletion analysis revealed that the His-Asn repeats and proline-rich regions were not necessary for DNA-binding and trans-activation. Although these classic functions of transcription factors are unaffected, the authors suggest regulation may occur through another mechanism such as protein modification or degradation.

7. Signaling molecules of the Fe deficiency response

While it is becoming increasingly clear that hormones and other small molecules such as nitric oxide (NO) play crucial roles in modulating gene expression and in altering root system architecture in response to changes in Fe availability, such control appears to be particularly complex: one hormone can affect the synthesis of another and there is likely to be cross talk among the intricate signaling pathways through which hormones work.

First, we will examine positive regulation of the Fe-deficiency response by auxin, ethylene, and NO, all of which are found at increased levels in Fe deficient conditions [73]. We will then review the data implicating both cytokinin and jasmonate as negative regulators of the Fe deficiency response [74, 75]. Refer to Table1 for a summary of what is known about the role of each hormone or signaling molecule in the strategy I Fe deficiency response and Figure 2 for a graphical model.

Table 1.

Summary of hormonal and small molecule signaling involvement in the strategy I Fe deficiency response.

Signal	Regulation on IRT1/FRO2	Does action depend on FIT?	Mechanistic detail	Proposed reason for link to Fe homeostasis	References
Auxin	Positive	Yes	Fe effects AUX-1	Modification of root architecture to meet nutrient demand	[77,78]
Ethylene	Positive	Yes	FIT interacts with EIN3/EIL1	Reduction of photooxidative damage	[42, 84, 85, 87]
Nitric oxide	Positive	Yes	NO stabilizes FIT		[43, 92, 93]
Cytokinin	Negative	No		Modification of root architecture to meet nutrient demand	[74]
Jasmonate	Negative	No		Stress adaptation i.e. tolerance to herbivores	[75]

Figure 2. Model of hormone and small molecule effects on regulation of the Strategy I Fe deficiency response.

Auxin, ethylene, and NO have been implicated as positive regulators of the Fe acquisition genes FIT, FRO2, and IRT1, while cytokinin and jasmonate have been demonstrated to negatively regulateFRO2 and IRT1 in a FIT-independent manner. Fe from the phloem is thought to serve as a negative regulator of Fe deficiency gene expression.

7.1. Positive regulators: auxin, ethylene, and NO

7.1.1 Auxin: Strategy I plants

Plant organ formation has been shown to depend on dynamic gradients of the hormone auxin [76]. Cross talk between auxin and Fe homeostasis had been suggested earlier with clear evidence that auxin plays a role in the root morphology response to Fe deficiency [77] but there have been some new important findings regarding the role of auxin in regulating the physiological response to Fe deficiency. Fe deficiency leads to increased auxin synthesis in Arabidopsis, which results in enhanced expression of FIT and FRO2 [34]. Exogenous auxin addition further stimulated transcription of these genes, while auxin inhibition decreased expression. Additionally, in an auxin overproducing mutant, yucca, FIT and FRO2 expression were increased in Fe deficiency compared to WT [34].

The most recent connection of auxin to the Fe deficiency response was the discovery that localized Fe supply regulates lateral root growth by affecting the AUX-1 transporter mediated auxin distribution [78]. Root system architecture, such as lateral root formation, is known to depend on nutrient availability [79]. The authors wanted to better understand how nutrient signals are integrated into developmental pathways by studying how Fe affects morphological root response [78]. Relative to homogenous Fe distribution in roots, localized Fe supply strongly induced lateral root growth. In low Fe conditions, lateral root growth was severely diminished in the irt1 mutant, but Fe addition to shoots could rescue this effect, suggesting that symplastic Fe is responsible for triggering local lateral root elongation. The Fe-induced root development effects depended on rootward auxin flow, with a critical role for AUX1, indicated by Fe-responsive AUX1 promoter activity and failure of mutants defective in auxin transport to exhibit lateral root elongation with localized Fe. The authors propose a model in which root uptake of local Fe is mediated by IRT1, which leads to accumulation of symplastic Fe. This pool of Fe can be replenished by Fe movement from the shoot or from other parts of the root and works to establish a local Fe gradient towards Fe deficient root tissues. The local symplastic Fe pool induces AUX1 expression and this facilitates auxin transport to further induce AUX1 expression and formation and elongation of lateral roots. This model demonstrates how hormone signaling can act with developmental pathways to allow for an increase in efficiency of micronutrient acquisition.

7.1.2. Auxin: Strategy II plants

As with all plants, optimal root architecture in rice is important for efficient nutrient uptake [79] and auxin has been demonstrated to mediate developmental processes in root architecture [73]. However, the mechanism for how root architecture is controlled is largely uncharacterized. A study by Qi et al. [80] demonstrates the involvement of OsARF12, an auxin response factor. OsARF12 is a transcription factor which can facilitate the expression of DR5-GFP, an auxin-inducible promoter construct [81]. osarf12 mutant rice plants have altered root architecture and are insensitive to auxin [80]. Additionally, in osarf12, Fe and other essential micronutrient content was lower than in WT and expression of OsIRT1 was reduced. These results indicate that OsARF12 plays a role in the crosstalk between the auxin response and Fe signaling. Understanding how root architecture is altered to maximize nutrient availability in plants is important for optimizing crop ability to cope with nutrient shortages.

7.1.3. Ethylene: strategy I plants

Ethylene, a gaseous plant hormone, has been implicated in a regulation of a diverse number of plant processes from seed germination to fruit ripening and senescence [82].

Several studies have indicated a physiological link between ethylene and Fe deficiency signaling. Upon Fe deficiency, ethylene is produced [83] and by affecting FIT or FER expression, ethylene can regulate the Fe-deficiency response in Arabidopsis and tomato [84, 85]. Treatment of Fe-deficient Arabidopsis or tomato plants with ACC (an ethylene precursor) enhanced expression of FIT or FER, and consequently the FRO and IRT genes. Conversely, treatment of plants grown in low Fe with ethylene inhibitors repressed expression of these genes. Because these effects are only seen in Fe-deficient conditions, as is the case with auxin, the authors propose a model in which ethylene is an activator for FIT or FER and Fe, possibly from the phloem, acts as an inhibitor.

Until very recently, ethylene signaling and the Fe deficiency response were only physiologically linked. Lingam et al. [42] uncovered the molecular basis of this observation by showing that ethylene signaling pathway transcription factors ETHYLENE INSENSITIVE3 and ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1) directly interact with FIT, are necessary for WT-level FIT accumulation, and are important for expression of FIT downstream target genes. In accordance with previous findings, they suggest that production of ethylene upon Fe deficiency is a positive signal for FIT regulation. Subsequently, by its physical interaction with EIN3/EIL1, the proteosomal degradation of FIT is reduced, which results in an increased level of Fe acquisition gene expression. According to their model, EIN3/EIL1 do not act with FIT to induce IRT1 and FRO2 expression, but rather serve to increase Fe acquisition by stabilizing FIT. The authors pose the question of why FIT interacts with EIN3/EIL1 specifically and in turn what is the function of EIN3/EIL1? The authors note that among the targets of EIN3/EIL1 in Fe-deficiency are shoot-expressed genes involved in remodeling of photosynthetic machinery and response to reactive oxygen species (ROS) [86]. This type of gene expression may reflect a response to photooxidative damage [87]. Although complete support is missing for this hypothesis, EIN3/EIL1 may function to reduce photooxidative damage in Fe deficient conditions and do so simply in part by increasing Fe uptake in roots by enhancing FIT accumulation [42].

7.1.4. Ethylene: strategy II plants

Recently, the first suggestion that ethylene is involved in Fe homeostasis in Strategy II plants comes from a study which demonstrates an involvement of ethylene in rice, the only graminaceous plant characterized which possesses a Strategy I-type system for Fe-regulation in addition to its standard Strategy II response [88]. Ethylene production of rice roots was increased when grown in Fe-deficient conditions and treatment with ACC conferred tolerance to Fe deficiency. By contrast, in another grass (barley), no induction of ethylene upon Fe deficiency or ethylene-mediated effects could be detected. Expression analysis showed that OsIRO2, OsNAS1, OsNAS2, OsYSL15, and OsIRT1 transcript abundance was increased, demonstrating that ethylene is involved in positive regulation of both Fe(II) and Fe(III)-PS uptake systems. Analysis of an RNAi line for OsIRO2 indicates that ethylene positively regulates this transcription factor to activate the expression of OsNAS1, OsNAS2, OsYSL15, and interestingly, OsIRT1. As mentioned previously, the central transcription factor upstream of OsIRO2 and OsIRT1 is OsIDEF1. Although OsIDEF1 is constitutively expressed [54], this study finds that OsIDEF1 expression is slightly induced by ACC, mainly in Fe-deficient conditions [88]. It remains to be determined whether this induction is relevant to the Fe deficiency response and tolerance to Fe deficiency. The authors also suggest that ethylene may directly regulate OsIRO2 and its downstream genes by interaction with other transcription factors such as EILS or EREBPs. They report that promoters of these genes contain ethylene cis-acting elements, supporting this hypothesis. If OsIRO2 indeed interacts with an EIL transcription factor as part of the involvement of ethylene in the regulation of the Fe deficiency response, this would be an analogous result to the recent interaction demonstrated between FIT and EIN3/EIL1 in Arabidopsis [42].

7.1.5. Nitric Oxide (NO)

The signaling molecule NO has been implicated in a huge array of plant processes related to development and response to abiotic and biotic stresses [89]. Like auxin, NO has a role in controlling lateral root development [90, 91]. However, NO does not usually appear to be the key player regulating these processes and more often seems to play a supportive role. Understanding how NO is connected to other hormonal networks may be a complicated task.

Like auxin and ethylene, NO is also proposed as an Fe-deficiency signaling molecule. In maize mutants defective in Fe uptake, NO can revert chlorosis [92]. In tomato, NO accumulation is triggered by Fe limitation [93]. As with auxin and ethylene, NO is a positive regulator of Fe uptake. Application of a NO donor to Fe-deficient tomato roots induces FRO1, IRT1, and FER, while NO scavenger treatment leads to repression of these same genes.

Very recently, NO has also been identified as a stabilizing stimulus for FIT protein abundance [43]. Application of NO inhibitors caused a decrease in FIT protein levels and FIT activity. This effect was reverse by using a proteasome inhibitor, supporting the hypothesis that NO works to decrease proteosomal degradation of FIT. At this point it is unknown if NO is involved in stabilizing and activating FIT directly or indirectly. The authors suggest that a direct role for NO could be the nitrosylation of Cys residues present in FIT. Indeed, S-nitrosylation of protein thiol residues has been shown to be an important mechanism for transduction of NO bioactivity [94-96]. It will be interesting to see if this modification occurs with FIT.

7.1.6. Interplay between auxin, ethylene, and NO

Because we know that an increase in auxin, ethylene, and NO all precede induction of Fe acquisition genes, the question that follows is which signaling molecule acts upstream of the other or whether they act in concert. Chen et al. [34] present a model in which, upon Fe deficiency, an increase in auxin acts upstream to induce NO which in turn acts to upregulate Fe acquisition machinery. Support for this model comes from the fact that FCR activity, NO level, and expression of FIT and FRO2 are increased in an auxin-over producing mutant while dramatically decreased in an auxin transport mutant. These results indicate that auxin acts upstream of NO.

As for ethylene and NO, recent work suggests these two signaling molecules work together to regulate the Fe-deficiency response. Garcia et al. [97] aimed to study the role of ethylene and NO in the expression of genes in Arabidopsis up-regulated in Fe deficiency beyond the three previously known genes; FIT, IRT1, and FRO2. By use of microarray analysis and RT-PCR to determine which genes are both upregulated by Fe deficiency, repressed by ethylene inhibitors, and responsive to NO, this study extends the role of ethylene to 16 new genes. Notably, this list includes known Fe homeostasis genes such as bHLH38, bHLH39, NAS1, and NAS2. It is clear from this work that NO and ethylene upregulate many of the same genes.

Garcia et al. [98] point out that previous studies demonstrate that NO enhances ethylene production, but they wondered if the converse was true. Indeed, they found this to be the case. Additionally, they show that the action of ethylene and NO in Fe homeostasis requires low Fe and thus Fe sufficiency serves as an inhibitory signal on these processes. They propose a model for Strategy I plants in which under Fe deficiency (and the absence of Fe likely from the phloem), roots increase production of ethylene and NO, with each one positively influencing the production of the other. They propose this activity generates an activating signal for the transcription of Fe acquisition genes.

7.2. Negative regulators

7.2.1. Cytokinin

Cytokinins, or CKs, are hormones which are known to control growth and developmental processes like cell proliferation, seed germination, and nutrient mobilization [99]. In addition to their negative effect on the Fe deficiency response, which we will discuss here, CKs have been shown to inhibit responses to nitrate, phosphate, and sulfate deficiencies in Arabidopsis [100-102].

CKs have the opposite effect as the positive regulators of the Fe deficiency response we have mentioned so far. Addition of exogenous cytokinin results in repression of IRT1, FRO2, and FIT which depends on cytokinin receptors [74]. To determine if FIT was responsible for mediating the CK-dependent downregulation of IRT1 and FRO2, IRT1 and FRO2 levels were examined in response to CK in WT and in the fit1 mutant. In WT, IRT1 mRNA is reduced by only two-fold in Fe-deficient conditions, while FRO2 is almost completely repressed. Both genes are further repressed with CK treatment. In fit-1, CK treatment strongly represses both IRT1 and FRO2, as in WT, suggesting that FIT is not necessary for CK-mediated IRT1 repression. Therefore, the CKs must act through a distinct signaling pathway from FIT.

CK treatment results in decreased primary root elongation [74]. Thus, it is suggested that altered root growth due to CK treatment may be a signal for the plant to limit root nutrient uptake due to the decrease in demand. Indeed, this paper shows that root growth inhibitory conditions, caused either by hormonal control or osmotic stress, lead to repression of IRT1 expression regardless of Fe supply. Therefore, the authors propose that CKs control root Fe uptake machinery in a growth-dependent manner to achieve a level of nutrient uptake that fits the demand of the plant.

7.2.2. Jasmonate

Jasmonates are oxylipin-based mobile plant hormones which act systemically and belong to a category of hormones called stress hormones that are known to act in response to various stimuli; i.e. wounding, instect attack, or UV light [103, 104].

Maurer et al. [75] showed that jasmonate is a negative regulator of Fe deficiency gene expression: FRO2, IRT1, and FIT were downregulated by application of methyl-jasmonate. Additionally, in mutants defective in jasmonate signaling activity, IRT1 and FRO2 mRNA levels were greater than in WT in Fe-deficient conditions. To determine if the jasmonate-mediated effects on the Fe-deficiency response depend on FIT, jasmonate treatments in the fit mutant were analyzed. In the mutant, though not to the degree as in WT, FRO2 and IRT1 are still partially induced in Fe-deficiency. However, upon jasmonate treatment, this partial induction is lost, with FRO2 and IRT1 gene expression at very low levels. These results show that, like with cytokinin, jasmonate inhibition of FRO2 and IRT1 is not dependent on FIT. The authors thus suggest that jasmonate serves as a subtle inhibitor for fine-tuning the Fe deficiency response, but does not systemically down-regulate Fe deficiency. Thus, the question arises as to what is the function of the jasmonate-mediated inhibition of Fe acquisition response. Since jasmonate belongs to the stress hormones, one possibility is that inhibition of acquisition mediated by jasmonate might be the result of a specific stress adaptation. One example given is the ability of jasmonate to confer tolerance to insect herbivores and necrotic pathogens. It is suggested that decreasing Fe uptake may aid to reduce cell death because necrosis involves Fe-dependent enzymes. As Maurer et al. mention [75], it is important to note that the positive regulators of Fe uptake, ethylene and NO, have been suggested as potential antagonists of jasmonate signaling [105, 106]. Clearly, the Fe deficiency response may be under control of complex interaction of hormones.

8. Conclusion

In the past several years, there has been significant progress in understanding how the Fe deficiency response in plants is controlled. Here we have described the key players of the major transcriptional networks that control Fe homeostasis in both grasses and non-grasses. It is evident that within these networks, there are multiple levels of control, i.e. transcriptional and post-transcriptional. A major goal in the field of Fe homeostasis in plants will be to uncover a potential master Fe sensor. We need to understand how the Fe status of a plant is sensed and how this signal is in turn relayed to the transcriptional networks controlling genes for Fe acquisition.

We have also summarized some of the major recent findings of how hormones and other signaling molecules are tied to the Fe deficiency pathway. Auxin, NO, ethylene, cytokinin, jasmonate and other signaling molecules not characterized or mentioned in this review may act in linear pathways and/or in a concerted manner. Further characterization and understanding of how these signals are involved in both the Fe deficiency response and other plant physiological processes will allow us to elucidate how they are integrated into a broader pathway.

The work of this field will help us achieve our overarching goals of improving plant growth and crop yields in less than optimal soils and addressing the problem of human Fe deficiency. We seek to achieve a food-based solution to this problem because it offers a sustainable approach to solving malnutrition.

• We summarize two strategies for Fe acquisition in plants.

• Strategy I response appears to be controlled by two transcriptional networks: FIT and POPEYE.

• Hormones are implicated in positive and negative control of the Fe deficiency response.