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Plant Cell. Dec 2004; 16(12): 3400–3412.
PMCID: PMC535881

The Essential Basic Helix-Loop-Helix Protein FIT1 Is Required for the Iron Deficiency Response

Abstract

Regulation of iron uptake is critical for plant survival. Although the activities responsible for reduction and transport of iron at the plant root surface have been described, the genes controlling these activities are largely unknown. We report the identification of the essential gene Fe-deficiency Induced Transcription Factor 1 (FIT1), which encodes a putative transcription factor that regulates iron uptake responses in Arabidopsis thaliana. Like the Fe(III) chelate reductase FRO2 and high affinity Fe(II) transporter IRT1, FIT1 mRNA is detected in the outer cell layers of the root and accumulates in response to iron deficiency. fit1 mutant plants are chlorotic and die as seedlings but can be rescued by the addition of supplemental iron, pointing to a defect in iron uptake. fit1 mutant plants accumulate less iron than wild-type plants in root and shoot tissues. Microarray analysis shows that expression of many (72 of 179) iron-regulated genes is dependent on FIT1. We demonstrate that FIT1 regulates FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation. We propose a new model for iron uptake in Arabidopsis where FRO2 and IRT1 are differentially regulated by FIT1.

INTRODUCTION

Iron is an essential element for most organisms, including plants, where it is required for cellular functions including photosynthesis and respiration. Iron deficiency poses an agricultural challenge because iron is one of the nutrients that most often limits plant growth. Iron deficiency also compromises human health because it is the leading human nutritional disorder worldwide, and plants are the most common source of dietary iron. Therefore, improving the iron content of plants will benefit both agriculture and human health. Engineering plants with increased iron levels requires an understanding of how plants cope with the challenges of acquiring iron from the soil and how these processes are controlled.

Plants overcome iron-deficient growth conditions in one of two ways. Nongraminaceous plants, including Arabidopsis thaliana, use the Strategy I response, which consists of the induction of three activities under low iron conditions (Römheld, 1987). A H+-ATPase extrudes protons into the rhizosphere to lower the pH of the soil, thus making Fe(III) more soluble. The inducible ferric chelate reductase activity of FRO2 reduces Fe(III) to Fe(II) (Robinson et al., 1999), which was recently shown to be the rate limiting step for iron acquisition from the soil (Connolly et al., 2003). Fe(II) is then transported into the plant by IRT1 (Eide et al., 1996), which is the major iron transporter of the plant root (Henriques et al., 2002; Varotto et al., 2002; Vert et al., 2002). The grasses (Takagi et al., 1984), as well as species of bacteria and fungi (Guerinot, 1994), use the Strategy II response, which relies on chelation of Fe(III) rather than reduction. Phytosiderophores are released into the soil where they chelate Fe(III) and are then internalized in the iron-bound state via specific transporters (Curie et al., 2001).

Reduction and transport of iron into the plant root are the final steps of iron acquisition in Strategy I plants. Transcripts of the genes responsible for these activities are themselves induced by iron deficiency, providing a primary level of regulation. FRO2 and IRT1 transcript levels are undetectable by RNA gel blot analysis when plants are grown under iron sufficient conditions but are greatly induced 24 h after transfer to iron-deficient medium (Connolly et al., 2002, 2003). Protein levels are also under tight control because both FRO2 and IRT1 are subject to posttranscriptional control (Connolly et al., 2002, 2003), signifying the importance of controlling the uptake of this essential yet potentially toxic metal. In addition to the local induction by iron, signals that induce iron deficiency responses include shoot-derived signals as shown by reciprocal grafting experiments (Grusak and Pezeshgi, 1996) and split-root experiments (Schmidt et al., 1996; Schikora and Schmidt, 2001; Vert et al., 2003). The nature of this shoot-derived signal is not yet understood. Iron transport, homeostasis, and signaling have been recently reviewed (Curie and Briat, 2003; Hell and Stephan, 2003).

To date, the only description of a putative transcription factor involved in iron acquisition in plants is that of the fer protein in tomato (Lycopersicon esculentum). The FER mutant was initially characterized as being unable to induce Strategy I responses under iron deficiency (Brown et al., 1971; Brown and Ambler, 1974). Grafting experiments have shown that the fer gene is required in roots but not in shoots (Brown et al., 1971). Recent cloning of the fer gene reveals that it encodes a basic helix-loop-helix (bHLH) putative transcription factor (Ling et al., 2002).

Using microarray analysis, we have identified a putative bHLH transcription factor, Fe-deficiency Induced Transcription Factor 1 (FIT1), which regulates iron deficiency responses in Arabidopsis. Of the 161 predicted bHLH proteins in Arabidopsis (Heim et al., 2003; Toledo-Ortiz et al., 2003), FIT1 is the closest homolog to the tomato fer gene. Both genes appear to play similar, but not identical, roles in their respective systems (Ling et al., 2002). The superfamily of bHLH transcription factors is conserved from yeast to mammals. bHLHs are the second largest transcription factor family in plants and govern a wide range of biological processes (Riechmann et al., 2000). The conserved bHLH domain consists of ~18 hydrophilic and basic amino acids comprising the basic region, which permits binding to DNA at the hexanucleotide E-box sequence 5′-CANNTG-3′. Two stretches of hydrophobic residues separated by a loop region form two amphipathic α-helices and allow these proteins to form homodimers and/or heterodimers (Voronova and Baltimore, 1990; Toledo-Ortiz et al., 2003).

Here, we report the characterization of the essential gene FIT1 and describe its role as it relates to iron deficiency responses in Arabidopsis. FIT1 is required for proper regulation of ferric chelate reductase activity and iron transport into the plant root. This is achieved by regulating the Fe(III) chelate reductase FRO2 at the level of steady state mRNA accumulation and by controlling protein accumulation of the Fe(II) transporter IRT1. FIT1 also controls many genes implicated in iron homeostasis as well as many novel genes, as we demonstrate by microarray analysis of a fit1 mutant.

RESULTS

Identification of FIT1

Microarray analysis was used to compare transcript abundance under varying iron conditions in wild-type and frd3 mutant plants to identify novel iron-regulated genes involved in the uptake and distribution of iron. frd3 plants exhibit constitutive iron deficiency responses independent of iron supply and may be defective in iron distribution or signaling (Rogers and Guerinot, 2002). The putative bHLH transcription factor FIT1 was among many genes identified whose message is iron regulated in wild-type roots and deregulated in the frd3 mutant (our unpublished data). To confirm the microarray data, RNA gel blot analysis was performed showing that FIT1 message is more highly expressed in iron-deficient roots than in iron-sufficient roots and is undetectable in shoots of wild-type plants (Figure 1A). In frd3 plants, levels of FIT1 message are equivalent regardless of iron supply in roots and absent in shoots by RNA gel blot analysis (data not shown).

Figure 1.
Steady State Levels of mRNA and Protein of Iron Uptake Genes in fit1-1 Plants.

FIT1 is a putative transcription factor, and predictions of its DNA recognition sequence can be made. The identity of nonconserved amino acids in the basic region of bHLH transcription factors determine the affinity for specific E-box sequences in the promoters of regulated genes, the most common being the G-box 5′-CACGTG-3′ (Robinson et al., 2000). Upon examination of specific residues in the bHLH domain, FIT1 is predicted to belong to a subgroup of bHLHs that recognize the E-box 5′-CANNTG-3′, but not the G-box in the promoters of target genes (Toledo-Ortiz et al., 2003).

FIT1 is the Arabidopsis bHLH that shares the most homology outside of the bHLH domain with the fer gene in tomato, which was recently cloned and described as playing a role in regulating iron uptake (Ling et al., 2002). The FER and FIT1 proteins share 42.5% identity and 72% similarity (Ling et al., 2002). Indeed, these genes appear to have related functions because mutations in these genes result in similar growth phenotypes in planta. However, there are some striking differences between these two genes, such as expression pattern, localization, and effect on expression of iron uptake genes, as we describe below.

Localization of FIT1 to Iron-Deficient Roots

To identify the regions within the root where FIT1 mRNA is expressed, transgenic plants expressing the β-glucuronidase (GUS) reporter gene fused to the 5′ end of FIT1 under control of the endogenous FIT1 promoter (FIT1-GUS) were analyzed for GUS staining. Seedlings from the four T3 transgenic lines examined showed GUS staining in the outer cell layers of the root as early as day 2 in plants germinated on iron-deficient plates (Figure 2A). GUS staining was observed in the differentiation zone but was absent from the elongation and meristematic zones of plants germinated on iron-deficient plates at days 2, 3, 4, 5, 7, and 9 post-germination (Figure 2B). Strong GUS staining was detected in the lateral roots of day 9 plants (Figure 2C) in a similar pattern as observed with the main root (Figure 2B). Staining of root hairs (Figure 2B) indicates FIT1 expression in the epidermis. When plants were germinated on iron-sufficient plates, very weak GUS staining was observed only at day 7 and day 9, primarily in the lateral roots (data not shown). To further localize FIT1 expression, RNA in situ hybridization was performed on longitudinal root sections. Hybridization of a FIT1 antisense probe to day 7 iron-deficient roots showed that FIT1 message is present in the outer cell layers (Figure 2D). No signal was observed in sections of day 7 iron deficient roots hybridized with a FIT1 sense probe (Figure 2E). No signal was observed when sections of iron-sufficient roots were hybridized with either the sense or antisense probes (data not shown). FIT1 message, like that of the Fe(III) chelate reductase FRO2 (Connolly et al., 2003) and Fe(II) transporter IRT1 (Vert et al., 2002), is localized to the outer cell layer of iron-deficient roots, making these iron deficiency response genes potential targets of FIT1 regulation.

Figure 2.
Localization of FIT1 to Iron-Deficient Roots.

FIT1 T-DNA Insertion Lines Indicate a Role for FIT1 in Iron Uptake

A T-DNA insertion line, fit1-1, was identified by a PCR-based screening approach from the Arabidopsis Knockout Facility's collection of 60,480 insertion lines (Krysan et al., 1999). A second T-DNA insertion line, fit1-2, was obtained from the Salk collection of insertion lines (Alonso et al., 2003). The T-DNA is inserted 106 bp upstream of the FIT1 start codon in the fit1-1 allele and 70 bp downstream of the start of the third exon in the fit1-2 allele. RNA gel blot analysis of wild-type Wassilewskija (Ws) and fit1-1 plants shows that FIT1 mRNA is greatly reduced in fit1-1 plants. In wild-type plants, FIT1 mRNA is abundant in iron-deficient roots, at low levels in iron-sufficient roots, and absent in shoots regardless of iron supply (Figure 1A). In the fit1-1 background, a very low amount of FIT1 mRNA is detectable only in iron-deficient roots (Figure 1A). RNA gel blot analysis of FIT1 in the wild-type Columbia and fit1-2 backgrounds also shows reduced transcript accumulation in the mutant, and this transcript appears slightly larger than that of the wild type (data not shown).

Because heterozygous fit1 insertion lines have no visible phenotype, the mutations in FIT1 are recessive loss-of-function alleles. Homozygous insertion lines of both fit1 alleles show a severe growth phenotype, indicating that FIT1 is essential for survival. Disruption of the FIT1 gene results in lethality at the seedling stage (Figure 3A). fit1-1 and fit1-2 seedlings are chlorotic, consistent with iron starvation, are smaller than their wild-type counterparts, and die 2 to 3 weeks post-germination. Watering fit1 plants with supplemental iron overcomes lethality and permits fit1 plants to reach the reproductive stage (Figure 3B), suggesting that FIT1 is required for iron uptake. A genomic fragment consisting of the FIT1 coding sequence and flanking 5′ and 3′ regions was used to complement the growth phenotype of both fit1 insertion alleles (fit1-1:FIT1 and fit1-2:FIT1) to show that lethality is a result of disruption of the FIT1 gene. Seedling lethality was completely reversed in all 11 independent fit1-1:FIT1 (Figure 3A) and fit1-2:FIT1 T2 transgenic lines. Although a few plants appeared slightly chlorotic compared with the wild type, all plants were much healthier than fit1 plants, indicating that disruption of FIT1 is responsible for the observed phenotypes.

Figure 3.
Growth Phenotype, Rescue, and Complementation of fit1-1 Plants.

Altered Iron Deficiency Responses in the fit1 Mutant Background

To determine if FIT1 regulates the iron deficiency response genes FRO2 and IRT1, transcripts of these genes were analyzed in the fit1-1 background by RNA gel blot analysis. FRO2 mRNA, which is detected in iron-deficient roots of wild-type plants, was not detectable in fit1-1 plants (Figure 1A), indicating that FIT1 directly or indirectly regulates FRO2 at the level of mRNA accumulation. The ferric chelate reductase activity of FRO2 was also measured using the ferrozine assay. Ferric chelate reductase activity, which is highly induced in iron-deficient roots of wild-type plants, was not induced under iron deficiency in fit1-1 plants (Figure 4). This indicates that FRO2 protein activity is abolished in fit1-1 plants, as expected from the lack of detectable FRO2 mRNA. On the other hand, IRT1 transcript was still detectable in the fit1-1 background by RNA gel blot analysis (Figure 1A). IRT1 protein levels were then examined using an IRT1-specific peptide antibody to determine if IRT1 protein abundance is affected in the fit1-1 background. IRT1 protein accumulates in iron-deficient roots of wild-type plants, but there was no detectable IRT1 protein in the roots of fit1-1 plants (Figure 1B). Therefore, FIT1 controls both iron deficiency responses: FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation.

Figure 4.
fit1-1 Plants Lack Inducible Fe(III) Chelate Reductase Activity.

Elemental Analysis of fit1-1 Mutant Plants

To determine if fit1 plants display altered iron accumulation, the iron content of two biological replicates of wild-type and fit1-1 mutant 15-d-old seedlings was measured by inductively coupled plasma–mass spectrometry. The roots of fit1-1 plants have an average of 43% less iron than the roots of wild-type plants when grown under standard B5 conditions, iron-deficient conditions, and iron-sufficient conditions (Figure 5). This result was most pronounced under standard B5 growth conditions where fit1-1 roots have 51% less iron than wild-type roots. Altered iron accumulation was also evident in the shoots, where fit1-1 plants have 42% less iron when grown under B5 conditions and 21% less iron when grown under iron-sufficient conditions compared with the wild type (Figure 5). Each of these differences was statistically significant. However, there was no significant difference in shoot iron content when plants were grown under iron-deficient conditions (Figure 5).

Figure 5.
Iron Content of Wild-Type and fit1-1 Plants.

35S:FIT1 Transgenic Plants Have No Obvious Phenotype

Because decreasing the amount of FIT1 mRNA has dramatic effects on plant survival, we wanted to determine if increasing FIT1 copy number has an effect on plant growth or the expression of iron deficiency response genes. Transgenic plants expressing FIT1 cDNA under control of the strong, constitutive 35S promoter were generated, and three independent homozygous single insertion lines were examined in the T4 generation. RNA gel blot analysis shows that FIT1 mRNA was highly expressed regardless of iron supply in the roots and shoots of all three transgenic lines (Figure 6A). The expression patterns of both FRO2 and IRT1 are unchanged compared with the wild type in the three 35S:FIT1 transgenic lines studied (Figure 6A). IRT1 protein accumulation was also unchanged in 35S:FIT1 plants compared with the wild type (Figure 6B). There was no significant difference in iron content between wild-type plants (average Fe ppm 77.2) and three independent 35S:FIT1 transgenic lines (average Fe ppm 79.0, 78.3, and 76.7), also supporting the conclusion that iron deficiency responses are not upregulated in 35S:FIT1 plants. 35S:FIT1 plants showed no obvious growth phenotype when grown on standard B5, iron-deficient, or iron-sufficient conditions or when grown on soil.

Figure 6.
Expression of Iron Uptake Genes in 35S:FIT1 Plants.

Identification of Genes under FIT1 Regulation by Microarray Analysis

Our expression analysis has identified FRO2 as a potential direct target of FIT1 regulation. To identify additional FIT1 targets, we performed microarray analysis to compare expression levels in the roots of wild-type and fit1-1 plants grown under iron-sufficient and iron-deficient conditions. Of particular interest are those genes that are iron regulated in the wild type and deregulated in the fit1-1 mutant. Both of these conditions were tested for statistical significance, and genes with a Bayesian P value < 0.05 were studied. Of the 179 genes that are twofold upregulated in response to iron deficiency in wild-type roots, 72 are also deregulated at least twofold in fit1-1 iron-deficient roots compared with wild-type iron-deficient roots. These 72 genes are grouped based on their regulation by FIT1 and on their predicted function. The signal intensities and fold changes in response to iron supply for the average of two replicates are presented in Table 1. Several genes in this group have previously been shown to be upregulated in response to iron deficiency, including IRT1 (Eide et al., 1996; Connolly et al., 2002), IRT2 (Vert et al., 2001; Wintz et al., 2003), NAS1 (Wintz et al., 2003), and NRAMP1 (Curie et al., 2000; Thomine et al., 2000).

Table 1.
Summary of Microarray Analysis

Most genes, 59 out of 72, showed a near complete loss of iron regulation in the fit1-1 mutant. We conclude that the increase in expression of these genes under iron deficiency in wild-type plants requires the presence of FIT1. Eight of the 72 transcripts retain partial iron regulation in fit1-1 plants. We propose that FIT1 is involved in the regulation of such genes but is not the only regulatory factor. A third category of genes are those whose expression level is elevated under iron-sufficient conditions in fit1-1 plants compared with the wild type. Five genes, including IRT1, fit this description. One explanation is that these genes are experiencing a relief from negative regulation in the fit1-1 mutant. FIT1 would normally activate a negative regulator, but in the absence of FIT1, this repression is removed and expression levels are elevated in fit1-1 plants. However, we favor the explanation that fit1-1 plants are experiencing iron deficiency, even when grown under iron-sufficient conditions, because of a defect in iron uptake. This is supported by our data that fit1-1 roots contain less iron than wild-type roots (Figure 5). Therefore, these genes may be responding to the iron-deficient conditions of the fit1-1 mutant rather than to loss of FIT1. IRT1 shows this expression pattern (elevated transcript levels in fit1-1 plants compared with the wild type under iron-sufficient conditions). IRT1 mRNA is known to be regulated by iron (Eide et al., 1996; Connolly et al., 2002; Vert et al., 2003), and we have shown that the IRT1 transcript still responds to iron deficiency in the fit1-1 mutant (Figure 1). Therefore, IRT1 would be predicted to fall into the category of genes responding to the iron-deficient conditions of the fit1-1 mutant rather than to loss of FIT1.

Notably, several genes that are known to be iron regulated are not affected by loss of FIT1. RNA gel blot analysis of NRAMP3 (Thomine et al., 2000) and recent microarray analysis of FRO3 (Wintz et al., 2003) have shown that these genes are upregulated in response to iron deficiency, yet these transcripts are not affected by loss of FIT1 (data not shown). Similarly, Ferritin1 and Ferritin4 have been shown to be downregulated in response to iron deficiency (Petit et al., 2001a; Wintz et al., 2003). Our analysis also demonstrates regulation of these genes by iron, but this expression pattern is independent of FIT1 (data not shown). These results demonstrate that only a portion of iron-regulated genes are under FIT1 regulation. FRO6 and FRO8 showed very low expression under iron-sufficient and iron-deficient conditions in wild-type and fit1-1 plants. FRO2, which is clearly iron regulated and deregulated in fit1-1 (Figure 1), is not represented on the ATH1 chip. The remaining four members of the FRO family in Arabidopsis are also not represented on the ATH1 chip.

It is possible that groups of genes with similar expression patterns may be controlled by the same regulator(s). We analyzed 1000 bp of sequence upstream of the translational start of selected FIT1-regulated genes. The promoter regions were searched for occurrences of the bHLH recognition sequence 5′-CANNTG-3′ to determine if such sequences are overrepresented compared with genome-wide noncoding sequences. We used the calculation that noncoding sequences in the Arabidopsis genome have a GC content of 33% to determine if these recognition sequences occurred more frequently than would be predicted by chance (Arabidopsis Genome Initiative, 2000). We examined the upstream regions of 10 transporters, seven transcription factors, and the 20 iron-regulated genes showing the greatest deregulation in fit1-1 from the list of 72 genes. We found that the bHLH recognition sequence was not significantly overrepresented in the upstream regions of the transcription factor group, the transporter group, or the top 20 genes showing the greatest deregulation in fit1-1. However, 35 of the 37 genes in this group contain at least one E-box in the 1000 bp of sequence upstream of the translational start (Figure 7).

Figure 7.
Promoter Analysis of Potential FIT1 Binding Sites.

DISCUSSION

Homozygous insertion alleles of fit1 are seedling lethal, but plants can be rescued by watering with supplemental iron (Figure 3A). A similar growth phenotype was previously reported for the irt1 mutant (Henriques et al., 2002; Varotto et al., 2002; Vert et al., 2002), suggesting that FIT1 may regulate IRT1 activity because the two mutants share the same growth phenotype. We show that fit1-1 plants are unable to induce Fe(III) chelate reductase and Fe(II) transport activities (Figures 1 and and4).4). fit1-1 plants accumulate less iron than their wild-type counterparts (Figure 5). However, when FIT1 copy number was increased in 35S:FIT1 transgenic plants, no obvious alteration in FRO2 or IRT1 expression or IRT1 protein accumulation was observed (Figure 6), and no growth phenotypes were revealed. Although we demonstrate that FIT1 message is being overexpressed in 35S:FIT1 plants, it is possible that FIT1 protein levels are not altered in the transgenic lines. Alternatively, overexpression of FIT1 alone may not be sufficient to alter expression of target genes. This is likely because bHLH transcription factors have been shown to dimerize with other bHLHs and with members of other transcription factor families, such as the MYB family (Goff et al., 1992; Abe et al., 1997; Grotewold et al., 2000), and both partners may be required to affect transcription of target genes. We identified several iron-regulated transcription factors representing a variety of families, including the bHLH and MYB families, by microarray analysis. We will investigate the possibility that FIT1 interacts with one or more of these potential binding partners to regulate iron uptake. However, because only one partner of the dimer needs to be regulated by iron, interacting partners involved in iron homeostasis may not yet be identified.

Because of sequence similarity outside of the bHLH motif, FIT1 is the Arabidopsis protein most closely related to the FER protein of tomato. As described here, the chlorotic and lethal phenotype of fit1 plants correlates with the description previously given for the fer mutant (Brown et al., 1971; Ling et al., 2002). We have shown that FIT1 message accumulates to higher levels under iron-deficient growth conditions than under iron-sufficient growth conditions in roots (Figure 1A). This is in contrast with the expression pattern of fer, which was reported to be independent of the iron supply, although like FIT1, fer is also expressed in a root-specific manner (Ling et al., 2002). fit1 and fer mutants both display an inability to induce ferric chelate reductase activity under iron deficiency. However, we have shown by RNA gel blot analysis that unlike FRO2, IRT1 message does accumulate in the fit1-1 mutant and that FIT1 controls IRT1 at the level of protein accumulation (Figure 1). This is in contrast with reports that Leirt1 mRNA abundance is dependent on the fer gene (Ling et al., 2002; Bereczky et al., 2003). The FIT1 and fer genes share similarities and differences in terms of their mRNA localization pattern. fer message was not detectable in the epidermal cells of the mature, root hair zone in tomato (Ling et al., 2002). We have shown in Arabidopsis that FIT1 localizes to root hairs in the differentiation zone by histochemical staining of FIT1-GUS transgenic plants (Figure 2B). Because transcripts of the iron deficiency response genes FRO2 and IRT1 localize to the outer layers of the root (Vert et al., 2002; Connolly et al., 2003), it is possible that FIT1 is regulating these activities in a more direct manner, whereas FER may govern these activities indirectly, perhaps through another regulator(s). We have demonstrated that FIT1 is expressed in the outer cell layers of the differentiation zone of wild-type roots (Figures 1 and and2).2). Our data is supported by large-scale microarray analysis (AtGenExpress, http://www.cbs.umn.edu/arabidopsis/) and expression analysis (Massively Parallel Signature Sequencing database, http://mpss.udel.edu/at/java.html), both indicating FIT1 expression is highest in roots compared with other tissues. Birnbaum et al. (2003) describe the localization of gene expression within the Arabidopsis root and report that FIT1 is more highly expressed in the outer cell layers (epidermis and lateral root cap) than in the stele, endodermis, and cortex (Birnbaum et al., 2003). Expression is also lowest in stage 1 (at the root tip) and highest in stage 3 (higher up the root where root hairs are present) (Birnbaum et al., 2003), similar to our results.

Previous studies in plants have identified promoter elements involved in the regulation by iron. The cis-regulatory element, iron-dependent regulatory sequence (IDRS), was identified in the promoter region of ZmFerritin1 (Petit et al., 2001b). The IDRS is conserved in AtFer1 and permits the induction of ferritin by iron by repressing ZmFer1 and AtFer1 when iron levels are low (Petit et al., 2001b; Tarantino et al., 2003). Although the IDRS has been shown to control genes that respond to the presence of iron, it is likely that a distinct mechanism exists for controlling genes that respond to iron deficiency. Recently, two iron deficiency–responsive elements (IDE1 and IDE2) were identified in the promoter of the IDS2 gene of barley (Hordeum vulgare) and shown to be required for iron deficiency–inducible expression of HvIDS2 in tobacco roots (Kobayashi et al., 2003). Several iron-inducible genes in barley, rice (Oryza sativa), and Arabidopsis contain sequences homologous to IDE1, including AtFRO2 and AtIRT1 (Kobayashi et al., 2003). Because FIT1 belongs to the bHLH family and is predicted to recognize a specific sequence in the promoters of regulated genes, we focused on the occurrence of the E-box motif in genes identified as potential FIT1 targets by microarray analysis. Although promoter analysis of the 10 transporters, seven transcription factors, and the 20 iron-regulated genes showing the greatest deregulation in fit1-1 revealed that the E-box sequence was not overrepresented in either of these three groups, it is still possible that FIT1 may be a direct regulator in most cases. Thirty-five of the 37 promoters analyzed, including that of FRO2, contain at least one E-box sequence (Figure 7), supporting the possibility that they are direct targets of FIT1. Because the E-box sequence will also occur at random, we do not anticipate that all E-boxes identified in Figure 7 will serve as FIT1 DNA binding sites.

Our analysis addresses plant iron deficiency responses on the scale of the whole genome using the ATH1 Affymetrix chip, which represents ~24,000 genes. There have been several microarray studies that have addressed iron deficiency induced changes in gene expression. However, these studies are difficult to use for direct comparison with our data because different experimental growth conditions were employed in each. For example, Thimm et al. used a 6000 cDNA chip and studied expression in older plants of a different ecotype (Landsberg erecta) that were grown hydroponically. They identified a set of genes induced under iron deficiency, including several encoding cytochrome P450-like proteins and two encoding zinc finger proteins (Thimm et al., 2001). Although we did not identify these exact genes in our microarray analysis, we also reported cytochrome P450 proteins and zinc finger transcription factors whose transcripts accumulate in response to iron deficiency. A chip representing 8987 rice clones was used to analyze genes responding to iron deficiency in barley roots (Negishi et al., 2002). The OsNAS1 gene and a zinc finger protein were identified in this way, which is consistent with our findings. In tomato, 1280 mineral nutrition-related genes were analyzed by microarray analysis, and LeIRT2, LeNAS, and a gene encoding a 14-3-3 protein were found to respond to Pi, K, and Fe deficiencies (Wang et al., 2002). As mentioned in Results, Wintz et al. reported IRT2 and NAS1 as being iron regulated using the Affymetrix DNA chip representing 8300 Arabidopsis genes (Wintz et al., 2003).

FRO2 and IRT1 messages are coordinately regulated in response to iron. In plants transferred from iron-sufficient to iron-deficient conditions, both transcripts are detectable by RNA gel blot within 24 h, with transcript levels peaking 3 d after transfer (Connolly et al., 2002, 2003). When plants grown under iron-deficient conditions are transferred to iron-sufficient conditions, FRO2 and IRT1 mRNA levels quickly decrease and are undetectable by RNA gel blot 24 h after transfer (Connolly et al., 2002, 2003). Because these two genes with related activities have such similar expression patterns, it seems likely that they may be activated by the same regulator. However, our findings that FIT1 regulates FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation suggests that regulation of FRO2 and IRT1 is more complicated than previously thought. Because fit1-1 mutants die at the seedling stage and they lack FRO2 mRNA and IRT1 protein, we can conclude that FIT1 plays a significant role in iron homeostasis. FRO2 has been demonstrated to serve as the rate limiting step in iron acquisition (Connolly et al., 2003). Thus, its ability to regulate FRO2 makes FIT1 an important factor in iron uptake. Although it is possible that FIT1 binds directly to the FRO2 promoter to regulate transcription, there is likely an additional regulatory step(s) between FIT1 and IRT1. We now need to determine what regulators of IRT1 exist downstream of FIT1. Previous studies have shown that IRT1 is subject to posttranscriptional control. Transgenic plants expressing IRT1 under control of the 35S promoter accumulate IRT1 mRNA in both roots and shoots under iron-sufficient and -deficient conditions, but IRT1 protein only accumulates in iron-deficient roots (Connolly et al., 2002). IRT1 protein could be controlled posttranslationally by ubiquitination and endocytosis. Ubiquitin-mediated protein turnover of ZRT1, a metal transporter belonging to the same family as IRT1, has been demonstrated in yeast and is dependent on a critical Lys residue in the variable loop region (Gitan and Eide, 2000). IRT1 also contains Lys residues in the analogous region, which could serve as ubiquitination sites to mediate protein degradation (Connolly et al., 2002). One explanation for the loss of IRT1 in fit1-1 plants is that FIT1 regulates a factor(s) involved in IRT1 protein turnover. A model depicting IRT1 protein regulation by FIT1 is presented in Figure 8. FIT1 may negatively regulate IRT1 protein turnover. In wild-type roots under iron-deficient conditions, IRT1 protein turnover could be inhibited by Gene X, which is positively regulated by FIT1. Therefore, in the fit1-1 mutant, Gene X is not activated by FIT1, so IRT1 protein turnover ensues.

Figure 8.
Model of IRT1 Protein Regulation by FIT1.

Finally, FIT1 itself is iron regulated, so we will need to look for other iron-regulated transcription factors that control FIT1 and may directly regulate IRT1. Upstream of FIT1, we would also expect to find an iron sensor that itself is not affected by iron status but can sense iron levels and communicate this message through the activation or repression of downstream targets.

METHODS

Identification of FIT1 Loss-of-Function Mutants

The FIT1 locus identifier is At2g28160 and was named AtbHLH029 as reported (Heim et al., 2003). A FIT1-specific primer 5′-CAACAATCTCGGTTACATCATCACTAGAA-3′ and a T-DNA–specific primer 5′-CATTTTATAATAACGCTGCGGACATCTAC-3′ were used to screen the Arabidopsis Knockout Facility's collection of T-DNA insertion lines (Ws ecotype) by PCR (Krysan et al., 1999). The fit1-1 allele was identified and confirmed by DNA gel blot hybridization. The insertion site was confirmed by DNA sequencing using the T-DNA–specific primer. The fit1-2 insertion mutant (Columbia-0 ecotype) was obtained from the Salk collection (Alonso et al., 2003). Both seed stocks were obtained from the ABRC (http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm).

Plant Materials and Growth Conditions

Seeds were surface sterilized in 95% ethanol followed by gentle shaking in 25% bleach/0.2% SDS for 20 min. Seeds suspended in 0.15% agar were placed in the dark at 4°C for 2 to 4 d, then plated on Gamborg's B5 medium (Sigma, St. Louis, MO) with 2% sucrose, 1 mM Mes, and 0.7% agar, pH 5.8. At the four- to six-true-leaf stage, seedlings were transferred to iron-sufficient plates containing 50 μM Fe(III)-EDTA or iron-deficient plates containing 300 μM ferrozine [3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine sulfonate] (HACH Chemical, Ames, IA) for 3 d. These media also contain macronutrients and micronutrients (Marschner et al., 1982), 0.7% agar and 1 mM Mes, pH 6.0. Plants were grown at 21°C under constant light (~90 μE·m−2·s−1) under a yellow filter (acrylic yellow-2208; Cadillac Plastic and Chemical, Pittsburgh, PA) to protect the Fe(III)-EDTA from photochemical degradation (Hangarter and Stasinopoulos, 1991). Plants used for in situ hybridization studies were germinated directly on iron-deficient or iron-sufficient plates and grown vertically. Plants used for GUS histochemical staining were germinated directly on iron-deficient or iron-sufficient plates. Ferric chelate reductase assays were performed as previously described (Yi and Guerinot, 1996). Pools of five plants were analyzed in triplicate, and standard deviations were calculated. Soil-grown fit1 plants were watered with 0.5 g/L of Sequestrene (Helena Chemical, Spartanburg, SC) two times per week during the seedling stage. At the reproductive stage, every other watering was supplemented with 0.5 g/L of Sequestrene.

Plasmid Construction and Plant Transformation

35S:FIT1 Fusion

The FIT1 cDNA, clone RZ108e05 (Asamizu et al., 2000), was subcloned into pGEM-TEasy (Promega, Madison, WI). The cDNA was excised using BamHI and cloned into the BamHI site of pCGN18. pCGN18 was the gift of T. Jack (Department of Biological Sciences, Dartmouth College, Hanover, NH; Connolly et al., 2002). The construct was moved into Agrobacterium tumefaciens strain ASE and transformed into wild-type Columbia plants. All plant transformations were done by the floral dip method (Clough and Bent, 1998).

FIT1-GUS Fusion

A 1924-bp PCR fragment was amplified from Ws genomic DNA using 5′-CGGGATCCCAACACCTAGATGGAATC-3′ and 5′-AACACTGCATCTCCAACAATCCATGC-3′ primers and subcloned into pGEM-T Easy (Promega). A fragment containing 1333 bp of sequence upstream of the FIT1 translational start and the 5′ 435 bp of coding sequence was excised by HindIII and BamHI digestion and cloned into pCAMBIA1381Xa (GenBank accession number AF234303) at the HindIII and BamHI sites creating an in-frame translational fusion to the gusA gene that was confirmed by DNA sequencing. The construct was moved into Agrobacterium strain GV3101 and transformed into wild-type Ws plants.

fit1 Complementation

A 2722-bp fragment containing the FIT1 coding sequence, 1140-bp sequence upstream of the translational start, and 386-bp downstream of the stop codon was amplified by PCR with engineered XbaI sites and cloned into the XbaI site of pCAMBIA1300 (GenBank accession number AF234296). The plasmid was moved into Agrobacterium strain GV3101 and transformed into fit1-1 and fit1-2 plants.

Gel Blot Hybridization

Root and shoot tissues were harvested from plants grown under iron-sufficient or iron-deficient conditions. Total RNA was prepared by hot phenol extraction and treated with glyoxal (McMaster and Carmichael, 1977). Ten micrograms of total RNA was separated on a 1.2% agarose gel in 10 mM NaPO4, transferred to a nylon membrane, and UV cross-linked. Hybridizations were performed at 42°C in 50% formamide according to standard procedures (Ausubel et al., 2004). Individual blots were probed with 32P-labeled FIT1, FRO2, or IRT1 cDNAs. Blots were visualized after 2 to 16 h exposure on a Typhoon Phosphorimager screen (Molecular Dynamics, Sunnyvale, CA).

Immunoblot Analysis

Immunoblot analysis was performed as previously described (Connolly et al., 2002). Briefly, total protein was prepared from plants grown under iron-sufficient or iron-deficient conditions as described above. Approximately 10 μg of protein was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were blocked, incubated overnight with affinity-purified IRT1 peptide antibody, washed, and incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase for 1 h. Chemiluminescence was performed using a Western Lightning kit (Perkin-Elmer, Boston, MA).

Elemental Analysis

Wild-type and fit1-1 mutant plants were grown on standard B5, iron-sufficient, or iron-deficient plates. Root and shoot tissues were harvested and dried overnight in a 65°C oven. Elemental analysis was performed using inductively coupled plasma spectroscopy at Purdue University as described (Lahner et al., 2003). Standard deviations were calculated for two biological replicates.

In Situ Hybridization

The FIT1 cDNA clone RZ108e05 (Asamizu et al., 2000) cloned into pBluescript II SK− at the EcoRI and XhoI sites (FIT1-SK) was used to generate sense and antisense probes for in situ hybridization. Probes were labeled with digoxigenin-11-UTP (Roche Diagnostics, Indianapolis, IN). Linearization of FIT1-SK was performed by ApaI digestion followed by transcription with T3 polymerase to obtain the sense probe. Linearization with SmaI followed by transcription with T7 polymerase was used to create the antisense probe. Tissue samples were fixed and embedded as described (Di Laurenzio et al., 1996), with the exception that after the 70% ethanol dehydration step, the tissue was embedded in 1% agarose. In situ hybridization was performed as previously described (Long et al., 1996; Long and Barton, 1998; Vert et al., 2002; Connolly et al., 2003).

GUS Histochemical Staining

GUS histochemical staining was performed on four independent T3 transgenic lines at day 4 and day 7 growth on iron-deficient and iron-sufficient plates containing hygromycin (25 mg/mL). One representative T3 line was examined under the same conditions at several time points between day 1 and day 9. Seedlings were incubated with the substrate 5-bromo-4-chloro-3-indolyl β-d-glucuronide as described (Jefferson et al., 1987).

Microarray Analysis

Total RNA from two biological replicates was reverse transcribed, labeled, and hybridized to individual ATH1 Affymetrix chips at the UCI DNA Array Core Facility (Irvine, CA). Expression data was analyzed using GeneTraffic (Iobion Informatics, La Jolla, CA). Determination of statistical significance was performed using the Cyber-T statistics program (http://visitor.ics.uci.edu/genex/cybert/).

Acknowledgments

The authors thank Brett Lahner and David Salt for performing elemental analysis, Carol Ringelberg for help with microarray analysis, Arijit Chakravarty for discussion of promoter analysis, Sara Thiebaud for experimental assistance, and Rob McClung for critical reading of the manuscript. This work was supported by a National Science Foundation Plant Genome grant (0077378-DBI) to M.L.G.

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Mary Lou Guerinot (ude.htuomtrad@tonireug).

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.024315.

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