Ironing out the issues: Integrated approaches to understanding iron homeostasis in plants

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Plant Science xxx (2013) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Plant Science

jo ur nal home p age: www.elsev ier .com/ locate /p lantsc i

eview

roning out the issues: Integrated approaches to understanding ironomeostasis in plants

ozalynne Samiraa,1, Anna Stallmanna,1, Lynnicia N. Massenburgb, Terri A. Longa,∗

Department of Plant Biology, North Carolina State University, Raleigh, NC 27695, USADepartment of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA

a r t i c l e i n f o

rticle history:eceived 27 February 2013eceived in revised form 7 June 2013ccepted 7 June 2013vailable online xxx

eywords:e homeostasis

a b s t r a c t

Plants initialize responses to environmental changes at all levels, from signaling to translation and beyond.Such is the case for fluctuations in the availability of iron (Fe), one of the most critical micronutrients forplants. The results of these responses are physiological and morphological changes that lead to increasediron uptake from the rhizosphere, and recycling and reallocation of Fe, which must be properly localizedwithin specific cells and cellular compartment for use. The use of reductionist approaches, in combinationwith in vivo and in situ Fe localization tools, has been able to shed light on critical signaling molecules,transcriptional regulators, transporters and other proteins involved in Fe homeostasis. Recent advances

e transporte translocationystems biologylemental analysis

in elemental distribution and speciation analysis now enable detection and measurement of Fe andother elements at resolutions never seen before. Moreover, increasing use of systems biology approachesprovide a substantially broader perspective of how Fe availability affects processes at many levels. Thisreview highlights the latest in vivo and in situ iron localization approaches and some of the recent advances

roteomicsetabolomics

redictive model

in understanding mechanisms that control Fe translocation. A broad perspective of how Fe localizationdata might one day be integrated with large-scale data to create models for Fe homeostasis is presented.

© 2013 Published by Elsevier Ireland Ltd.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. In vivo and in situ technologies to visualize and quantify Fe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Histochemical staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Transmission electron microscopy–electron spectroscopic imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Energy-dispersive X-ray analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Particle induced X-ray emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5. Synchrotron X-ray fluorescence microtomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.6. RPE, a turn-on fluorescent Fe3+ sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.7. Secondary ionization mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.8. Laser ablation inductively coupled plasma mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3. Application of Fe localization tools: Recent advances in understanding Fe translocation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1. Vascular uptake and translocation to the shoot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Xylem unloading and Fe translocation to seeds and beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Practical application of elemental analysis: A look ahead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Understanding Fe homeostasis in the–omics era . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1. Building the Fe deficiency transcriptional regulatory network. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: R. Samira, et al., Ironing out the issues:

Plant Sci. (2013), http://dx.doi.org/10.1016/j.plantsci.2013.06.004

4.2. Posttranscriptional and posttranslational response to Fe deficienc4.3. Metabolic responses to Fe deficiency: Shedding new light on Fe li

5. Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +1 919 515 0478.E-mail address: [email protected] (T.A. Long).

1 These authors contributed equally to this work.

168-9452/$ – see front matter © 2013 Published by Elsevier Ireland Ltd.ttp://dx.doi.org/10.1016/j.plantsci.2013.06.004

Integrated approaches to understanding iron homeostasis in plants,

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R. Samira et al. / Plant

. Introduction

Iron (Fe) is a critical nutrient for plants, essential for respira-ion, photosynthesis, and a number of other required functions. Feioavailability is determined, in part, by proper uptake and local-

zation throughout various organelles and cell types. Thus, theegulation of mechanisms involved in these processes is tightlyontrolled. Additionally, redox reactivity between Fe2+ and Fe3+

xidation states make Fe a useful cofactor in respiration and photo-ynthesis, yet capable of creating damaging reactive oxygen speciesf not chelated or stored. Based on these realities, the total soil orlant Fe content is not particularly informative without considering

ts more precise location and state.Until recently, most Fe homeostasis studies describe gene

egulatory processes, the activity of Fe transport mechanismsnvolved in the root, and the eventual use of Fe in specific cells andrganelles, such as leaf chloroplasts. A number of newer studiesre shedding more light on how Fe is translocation throughout thelant, and on how it is localized and reallocated between cellularompartments, cells and organs in response to Fe deficiency. How-ver, there is still much that we do not know about Fe homeostasisnd response to Fe deficiency, and current knowledge about Feomeostasis mechanisms is limited by the nature of available tools

n plant research. The ability to determine the mechanisms of Feovement (intracellular and intercellular) and use throughout

plant hinges on the ability to differentiate between various Feources and pools, and to visualize Fe localization in vivo and in situ.ecent advances in imaging techniques could provide momentum

or this field and will allow for a more broad and quantitativenderstanding of Fe pathways.

An increase in the use of systems biology approaches, or these of large-scale high-resolution transcriptomics, proteomics,etabolomics data to create predictive models [1], is also set to

ubstantially enhance our understanding of Fe homeostasis andeveal emergent properties that lead to proper localization andvailability of Fe. Today, whole genome transcriptional profilesxist for a wide range of species, for wild type and Fe homeostasisutants, for single time points, for whole roots across multiple

ime points, for specific root developmental zones, and evenor specific cell types [2–7]. Proteomic profiles for Arabidopsishaliana (Arabidopsis), tomato, cucumber, sugar beet, and Med-cago truncatula, and metabolomics profiles for a number of speciesre all revealing system-wide responses to Fe deprivation thatight even be conserved in animals [8,9]. How are plants able

o integrate these responses throughout the system? How doranscriptional, posttranscriptional and metabolic responses leado increased Fe concentration in specific tissues, cells, and cellularompartments? These and many other questions are yet to beompletely answered. However, a more holistic understanding ofe homeostasis is now emerging (Fig. 1). Excellent comprehensiveeviews of Fe homeostasis are found elsewhere [10–14]. Here, theatest in vivo and in situ Fe localization technologies are discussed

ith a focus on their current use and future utility. Additionally,ecent findings about Fe localization and what controls localizationre presented along with discussion of how we can advance thisnowledge by better integration of Fe localization tools withystems biology approaches.

. In vivo and in situ technologies to visualize and quantifye

Please cite this article in press as: R. Samira, et al., Ironing out the issues:Plant Sci. (2013), http://dx.doi.org/10.1016/j.plantsci.2013.06.004

The mechanisms that regulate physiological and molecularesponses to Fe deficiency all ultimately lead to the proper local-zation and availability of Fe within specific organs, cells andrganelles. The diverse set of pathways controlling Fe acquisition

PRESSe xxx (2013) xxx– xxx

and localization are difficult to resolve without spatial andtemporal understanding. Recent imaging advances that enablehigh-resolution detection, and, in some cases, measurement, of ele-mental concentration in vivo and in situ are presented. While adetailed review of many of these techniques is presented elsewhere[15], we describe and compare methods that have been utilizedto advance knowledge of Fe homeostasis (Table 1). Unless other-wise indicated most methods described below require the use ofinstrumentation often available, with moderate training, at manyresearch institutions.

2.1. Histochemical staining

Perls staining is one of the oldest methods for visualizing Fein plants. Briefly, in the presence of acid and potassium ferro-cyanide, acid hydrolysis releases ferric Fe from tissues, which canthen react with ferrocyanide ions, creating an insoluble, immo-bile, and visible potassium Fe–ferrocyanide complex known asPrussian blue [16]. Because of its specificity, low-cost, and sim-plicity, this method has been widely used in plant samples tovisualize ferric Fe. The use of this technique has been somewhatlimited due to poor sensitivity and penetration of ferrocyanide inhydrophobic tissues, making embryos with starchy endospermssuch as wheat grains difficult to stain [16]. Perls staining is also dif-ficult to detect in heavily pigmented plant samples, such as seedcoats [17]. Perls/diaminobenzidine (DAB) staining was recentlydeveloped to overcome these issues. In the presence of hydro-gen peroxide and DAB, the redox active Perls complex causesDAB to undergo oxidative polymerization, forming a brown col-ored pigment [16]. Implementation of tissue fixation and thinsectioning along with Perls–DAB stain can overcome the abovestated resolution and penetration problems, increasing resolu-tion and sensitivity [18]. Perls/DAB staining is also inexpensiveand does not require specialized instrumentation or expertise,making it one of the most popular in situ methods for detect-ing Fe in plants today. However, this technique does not enablequantification of elements and requires fixation for higher resolu-tion.

2.2. Transmission electron microscopy–electron spectroscopicimaging

Transmission electron microscopy (TEM) is a technique inwhich the differential interaction of a beam of electrons withan ultra-thin section of a specimen forms an image. Electronspectroscopic imaging (ESI) couples an electron energy loss spec-trometer with a conventional transmission electron microscopeto conduct quantitative imaging of the elements within thespecimen directly. Transmission electron microscopy–electronspectroscopic imaging takes advantage of the fact that when anelectron beam hits the specimen, the dwelling time, loss of energyand scattering of the beam will be different depending on theelemental composition of the specimen [19]. This method hasbeen used to create a 2-dimensional representation of organelleposition in relation to Fe dispersion [20], resulting in high res-olution in situ elemental maps. A downside to this method isthat thin sectioning and thus destructive sample manipulation isrequired.

2.3. Energy-dispersive X-ray analysis


Energy-dispersive X-ray analysis (EDX or EDAX) is based onthe principle that each element has a unique atomic structureresulting in a distinct and detectible emitted X-ray spectrum whenbombarded with high-energy electron beams [21]. It has beenused to detect Fe in thin embryonic sections of Avena sativa and

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Fig. 1. Examples of how proper localization of Fe (purple) requires control at many different levels, although much is still unknown (?). Interaction with ethylene-regulatedtranscription factors Ethylene Insensitive3 (EIN3) and EIN3-LIKE1 (EIL1), and possibly ubiquitination [68,69] control FER-Like Iron Deficiency Induced Transcription Factor(FIT) stability. Iron-Regulated Transporter 1 (IRT1) is transcriptionally repressed by an unknown repressor susceptible to rapid turnover [58], yet transcriptionally activatedby FIT, which forms heterodimers with related bHLH proteins (BHLH38 and BHLH39) [56]. IRT transports Fe into the epidermal cells, but it can be post-translationallymodified by polyubiquitination and targeted for degradation in the vacuole (V) [67]. NAS4 (Nicotianamine Synthase 4) is transcriptionally repressed by PYE, which may formhetermodimers with homologs (PYE-like (PPL)) that may be degraded by Fe deficiency-responsive BRUTUS (BTS) [3]. NAS proteins synthesize nicotianamine from SAM, ap privatr

Ctpc[tb

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roduct of the methionine cycle. Proteins involved in this cycle increases under Fe deed dots represent ubiquitination.

asuarina littoralis embryos [22]. More recently it was utilizedo detect decreased Fe–phosphate complexes in the vacuoles of



hosphate starved Arabidopsis plants, likely due to decreased Fehelation to phosphate and subsequent transport into chloroplasts23]. Similar to transmission electron microscopy–electron spec-roscopic imaging, fixation is required for this method and Fe cane detected at subcellular resolution.

ion [60]. NA2+–Fe is transported from root to shoot [36]. Gray indicates degradation,

2.4. Particle induced X-ray emission


When using particle induced X-ray emission (PIXE), elementsare exposed to ion beams (usually protons) that emit X-ray elec-tromagnetic radiation specific to the element of interest. PIXE isa non-destructive technique used to identify and quantify Fe intissues at the surface or near-surface layers [24]. Fixation of dry

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Table 1Comparison of elemental analysis techniques currently utilized to detect Fe in plants. References for examples of usage are indicated.

Technique Detectionmethod/resolutiona

Advantages Disadvantages Findings

Perls/DAB Light microscopyimage/subcellular

Facile Fixation required for highestresolution

Fe distribution duringembryogenesis, mature seeds,germination, and throughoutdevelopment [18]

Does not require expensiveinstrumentation ortechnical expertise

Quantification not possible Role of many Fe homeostasisgenes

Transmission electronmicroscopy/Electronspectroscopic imaging

Electron energy lossspectra/subcellular

High resolution Requires fixation Role of NRAMP3/4 [20]Role of pH in seed ferritindisassociation [51]

Energy-dispersive X-rayanalysis

X-rays/subcellular High resolution Requires fixation Fe–phosphate complexes invacuoles [23]Fe mislocalization inchloronerva [75]

Micro-particle induced X-rayemission

X-rays/subcellular Does not require fixation ofdry samples

Wet samples must be treatedwith cryo-fixation or chemicalfixation

Fe in bean seeds [17]

3D elemental mapconstruction

Nuclear localization of Fe [26]

Synchrotron X-rayfluorescence microtomography

X-ray fluorescence/cellular Does not require fixation ofdry samples

Instrument inaccessibility andexpertise

Role of VIT1, YSL1 and YSL3 inFe localization [45,50]

3D elemental mapconstruction

Turn-on fluorescent Fe3+ sensor:RPE

Fluorophore/N/A In vivo imaging capability Not yet used in plants N/A

Secondary ionization massspectrometry

Secondary ions/cellular High sensitivity Instrument inaccessibility andexpertise

Affect of Fe plaques on Asabsorption in rice roots [34]

Laser ablation inductively coupled ICP-MS/cellular High sensitivity Sample ablated Fe detection in tobacco cells

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plasma mass spectrometry(LA–ICP-MS)

a Resolution in published plant studies.

issue is not required prior to analysis, which reduces the event ofetal leaching during fixative soak. However, a wet sample must

e treated either by cryofixation or chemical fixation to prepare formaging and maintain sample integrity [25]. Recently developed

icro-PIXE (in which tightly focused beams are utilized for micro-copic detection of elements) substantially increased resolution ofhis technique, allowing for subcellular visualization [25]. Micro-IXE was useful in imaging Fe in pea (Pisum sativum) embryos [26]nd bean embryos [17].

.5. Synchrotron X-ray fluorescence microtomography

Synchrotron X-ray fluorescence (SXRF) visualized with micro-omography relies upon the unique absorption and fluorescenceharacteristics of elements exposed to synchrotron-based X-rayso create high-resolution 3D elemental maps [27]. Elementalistribution maps can be created from image data to obtain semi-uantitative metal concentrations. This method is non-invasive,nd particularly sensitive due to the high energy, tunability andeak scattering of the synchrotron-based X-ray beams [27]. How-

ver, since the process requires use of beam time on a synchrotron,ost to travel to and use a facility can be prohibitive. Seeds are aavorable choice for X-ray analysis, based on seed stability due toheir low water content [28]. However, recent improvements inample preparation, detection speeds, and data acquisition havenabled detection of nickel and zinc in hydrated cowpea roots [29],ncreasing the likelihood of using this method to detect Fe in freshlant samples in the near future.

.6. RPE, a turn-on fluorescent Fe3+ sensor


Nonspecific fluorescent probes for ions are commercially avail-ble, and can be used to detect ions and ion movement in vivon plants [30]. For example, use of the Fe2+-sensitive fluorophore,hen Green SK, was used to show that ferrous Fe movement

[35]

into the chloroplast is due to a uniport-mechanism caused bythe membrane potential of the inner envelope [31]. Recently,a highly sensitive, selective, and reversible turn-on fluorescentFe3+ sensor was developed to detect intracellular Fe3+ levelsand sub-cellular Fe3+ pools in human live neuronal cells [32]. Inthis technique methyl 6-(3′,6′-bis(diethylamino)-3-oxospiro-[iso-indoline-1,9′-xanthen]-2-yl)picolinate (RPE) was incubated withcells for 25 min allowing visualization of the Fe3+ pool with confo-cal microscopy. One advantage of this technique is that the visiblesignal disappears when tissue is treated with an Fe chelator, allow-ing for facile study of Fe dynamics without the need for expertiseand expensive detection equipment beyond a fluorescence micro-scope. As Fe localization in plants is associated with its transitionand chelation state, the possibility of using this reagent in plant cellsfor detecting Fe3+ levels, transition, and sub-cellular concentrationis particularly exciting.

2.7. Secondary ionization mass spectrometry

Secondary ionization mass spectrometry (SIMS) is a method inwhich an accelerated primary ion beam is used to bombard a sam-ple surface, generating secondary ions that are detected by a massspectrometer [33]. SIMS is utilized primarily on hard samples, in thefields of geology or material sciences, with optimal resolving powerof 1 �m. However, the more recent development of NanoSIMStechnology by CAMECA (http://www.cameca.com/) allows resolu-tion, and sensitivity between 50 and 100 nm, enabling cellular andsubcellular resolution, high sensitivity, and high mass resolutionanalysis of samples after ultrarapid cryofixation. NanoSIMS wasrecently used to determine how Fe plaques (Fe oxide/hydroxides


precipitates formed by the oxidation of ferrous Fe to ferric Fe)affect arsenic absorption in rice roots [34]. Use of this methodin the area of Fe research is hindered due to rarity of availableNanoSIMS instruments and the requirement for extensive train-ing.

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.8. Laser ablation inductively coupled plasma mass spectrometry

With laser ablation inductively coupled plasma mass spectrom-try (LA–ICP-MS) a sample area is scanned with a focused laseream according to a specified raster area, as low as 5 �m. Theelected area is evaporated by the laser beam and transported forCP-MS analysis by a carrier gas such as Argon [15]. This methodas been used to detect Fe and other metals in tree bark and seeds,et there are few examples of use of this method on fresh plantamples because laser ablation takes place under conditions whereehydration may occur, affecting metal distribution and sample

ntegrity [15]. Cryo-sectioning facilitates the use of this methodnd allowed researchers to produce a 2D image of Fe distributionnd other metals in fixed tobacco roots, shoots and leaves [35].

. Application of Fe localization tools: Recent advances innderstanding Fe translocation

Like most minerals, Fe is obtained primarily from the rhizo-phere, where it is absorbed first into the root epidermis. A numberf kinetic studies indicate that while in the epidermal symplast Fe isikely chelated to the nonproteinogenic amino acid nicotianamineNA) [36,37]. Fe travels symplastically through the cortex and endo-ermis, into the outer layer of the vasculature (the pericycle), andnally into xylem parenchyma cells before entering the apoplastnd being loaded into xylem cells for translocation and utilizationn the shoot.

.1. Vascular uptake and translocation to the shoot

Using an integrated mass spectrometry approach, researchersere able to determine that once loaded into the xylem from the

poplast, Fe is predominantly found as Fe3+-citrate [38]. Thus, whilee travels symplastically from the epidermis to the vasculatures Fe2+-NA, a ligand exchange is required for localization in theylem. In Arabidopsis, Ferric Reductase Defective (FRD3), a memberf the multidrug and toxin efflux family of transmembrane effluxroteins, is thought to mediate citrate efflux from the root per-

cycle and xylem parenchyma cells, while efflux of Fe itself intohe xylem is likely facilitated by the Fe transporter, Feroportin-1FPN1) [39,40]. frd3 mutants are chlorotic, exhibit constitutive Feeficiency responses, and accumulate a range of metals [41,42].legant work with Perls/DAB staining on frd3 mutants has recentlyhown striking accumulation of Fe deposits surrounding pericycle,ylem, and phloem cells, indicating that FRD3-mediated solubi-ization of Fe is required for xylem loading [39]. Once loadednto the xylem, Fe3+-citrate is translocated from the root to thehoot via the transpiration stream. Perls/DAB staining revealedhat Fe3+-citrate may be unloaded into the shoot apoplast byRD3 [39]. This is likely a conserved function as the expressionf soybean GmFRD3b was also implicated as a major contribut-ng factor to differences in Fe efficiency between soybean cultivars43].

Fe in the phloem is found primarily as Fe2+-NA [36,37]. However,he mechanism by which Fe3+-citrate is converted back to Fe2+-NAnd localized in the phloem is still an open question. Apoplastice3+-citrate is likely disassociated, allowing Fe3+ to be reduced toe2+ and then chelated to NA in the apoplast before uptake into thehloem. Analysis of quadruple nicotianamine synthase (nas4x-2)utants that do not synthesize NA revealed that lack of NA causes



rowth inhibition and chlorosis in young leaves but not in older andenescing leaves, and that there is an increase in FRD3 expressionnd citrate content in xylem sap in nas4x-2 mutants [44]. Addi-ionally, both Perls staining of whole leaves and atomic absorptionpectroscopy of leaf extracts indicate that frd3 nas4x-2 quintuple

PRESSe xxx (2013) xxx– xxx 5

mutants contain less Fe in older and senescing leaves than nas4x-2mutants [44]. These and other data suggest that while NA operatesprimarily in young leaves, FRD3-mediated citrate loading into theapoplast facilitates Fe transport to aging leaves. Moreover, citrateand NA may play partially redundant roles in the translocation ofFe from root to shoot [44].

Members of the Yellow Stripe Like (YSL) family of transportersfacilitate the movement of the nicotianamine–Fe complex fromapoplast to symplast in the shoot following xylem unloading[45–48]. Thus, it is likely that FRD3 facilitates citrate extrusion intothe apoplast of maturing leaves, which, by an unknown mecha-nism, facilitates Fe2+-NA uptake into phloem via YSL1 and YSL3proteins, allowing for translocation into reproductive tissues. Theuse of turn-on fluorescent Fe3+ sensors, such as RPE, could begin toanswer questions about how ligand exchange between the xylemand phloem is mediated and help explain how pH or specific Fereductases play a role in ligand exchange. Moreover, further studyof the interplay between citrate and NA, using metabolic profiling,could provide new insight into how Fe is mobilized within long andshort distance pathways, and how citrate could compensate for lossof NA.

3.2. Xylem unloading and Fe translocation to seeds and beyond

Perls/DAB staining has been used to show that in addition to itsrole in Fe3+-citrate xylem loading and unloading, FRD3 also playsa role in solubilizing and loading Fe3+-citrate into pollen, devel-oping embryos, and germinating seedlings. Without FRD3, citrateexcretion is decreased, insoluble Fe deposits are found outsidedeveloping pollen, and exogenous citrate is required for properseed germination [39]. The likelihood that FRD3-mediated citrateexcretion solubilizes Fe in the apoplast for uptake into various tis-sues throughout the plant is exciting, leading to new questionsabout how Fe3+ is reduced in the apoplast, what the predomi-nant forms of Fe and Fe ligands in pollen and developing embryosare, and what transporters facilitate the movement of Fe intoembryos and pollen. For example, while grafting experiments withysl1ysl3 double mutants indicate YSL1 and YSL3 are both requiredfor seed formation, synchrotron X-ray fluorescence microtomog-raphy confirms that Fe, while not mislocalized, is decreased indouble mutants. This result prompted researchers to suggest thatYSL1/3 proteins play an essential role in the short-distance translo-cation of Fe2+-NA from cauline leaves and inflorescence stems intoseeds [45]. Notably, YSL proteins are members of the oligopeptidetransporter (OPT) family. OPT3 has been shown to play a role inlong distance movement of Fe, as Perl staining revealed substan-tial build up of Fe3+ in opt3 leaves causing necrosis [49]. Althoughseeds from opt3 mutants were viable and germinated similar towild type, Fe content was reduced and seedlings germinated in Fedeficient growth media were chlorotic. This finding suggests thatalthough OPT3 affects seed Fe uptake from senescing plants, othertransporters are involved in Fe uptake from growth media upongermination [49].

Where is Fe localized during embryo development and at seedmaturity and how is it remobilized from seed stores during germi-nation? These questions have huge implications, as remobilizationof Fe from seed stores plays a critical role in seed germinationacross species. Moreover, seeds are an important Fe source in thehuman diet. Perls/DAB staining of Arabidopsis seeds reveals Fe inall cells during early stages of embryogenesis, yet as seeds reachmaturity Fe is localized primarily in the endodermal vacuolar com-


partment [16]. Studies utilizing synchrotron X-ray fluorescencemicrotomography and Perls/DAB staining indicate that VacuolarIron Transporter 1 (VIT1) facilitates Fe movement into vacuoles,and that without VIT1 Fe is still localized to vacuoles but detectedprimarily in cells external to the endodermal cells in the radical,

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ypocotyl, and cotyledons [16,50]. vit1 seeds are viable, and Feoncentration, germination, and growth are indistinguishable fromild type under normal growing conditions. However, disrup-

ion of Fe location in vit1 mutants inhibits Fe availability undere deficient conditions, affecting growth after germination [50].dditionally, use of transmission electron microscopy–electronpectroscopic imaging (TEM–ESI) on mutant seeds in which Naturalesistance Associated Macrophage Proteins (NRAMP) transporters areisrupted, namely nramp3nramp4, provided the high-resolution

mages needed to conclude that these transporters, similar to VIT1,re required to mobilize Fe from vacuolar stores to seeds duringermination, specifically under Fe deficiency [20]. Likewise, Micro-IXE along with Perls staining was used to show that, dependingn the genotype, the cells surrounding the provascular cells are

primary source of Fe during germination of Phaseolus seeds17].

In addition to the vacuoles, Fe is also stored in ferritin cages ineed amyloplast. TEM was used to compare the Fe storage capacityf ferritin in wild type and mutant pea lines in which the phyto-erritin specific domain, or “extension peptide,” was deleted [51].nlike animal ferritin, the association of phytoferritin subunits is

acilitated by low pH and this association results in long term Fetorage in plant seeds. During seed germination, the phytoferritinxtension peptide facilitates auto degradation, causing disassocia-ion and consequently release of Fe from the phytoferritin cavity inlastids [51]. Thus, Fe is released from multiple compartments dur-

ng germination although, once germinated, seedlings then acquiree from the rhizosphere.

A recent study has indicated that there are also checks andalances that prohibit excessive accumulation of free Fe duringermination. YSL4/6 may play a role in this process. Comparedo wild type, growth of ysl4ysl6 seedlings is impaired immedi-tely after germination on Fe deficient media, and YSL4/6 proteinccumulates in mature seeds yet decreases upon germination, sim-lar to the seed ferritin protein, FER2 [52]. Why? Since embryonichotosynthesis stops at seed maturity, excess Fe may be releasedrom the photosynthetic machinery by the activity of YSL4/6 andequestered by FER2 [52]. Since YSL6 is strongly expressed inenescing leaves and Fe accumulates in the chloroplast of senesc-ng leaves in ysl4/ysl6 mutants according to Perls/DAB staining,t is likely that this mechanism is mirrored in senescing leaves,

here photosynthesis decreases due to chloroplast degradation52].

.3. Practical application of elemental analysis: A look ahead

Due to ease of use, Perls/DAB staining is the most commonlysed method for cellular and subcellular detection of Fe. Since thisethod has led to significant advances in our understanding of Fe

ocalization mechanisms, why use other methods? Based on oururrent understanding of Fe homeostasis, Fe pools are dynamic,hanging rapidly in response to Fe availability. Thus, to gain morensight into how plants uptake, translocate, and reallocate Fe, Fe

ust be quantified and visualized with high accuracy at the cellu-ar and subcellular resolution. Elemental analysis techniques suchs micro-PIXE, synchrotron X-ray fluorescence microtomography,nd NanoSIMS allow for such measurements and, in some cases,he construction of 3D images of metal content (Table 1). There-ore, the challenges that lie ahead for researchers are to learn


ew methods for elemental analysis, collaborate with chemistsnd bioengineers to optimize the detection of elements such ase in plant tissues in vivo, and, finally, to work with mathemati-ians and systems biologists to create models for how the molecularesponses to Fe deficiency lead to dynamic changes in Fe movementn plants.

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4. Understanding Fe homeostasis in the–omics era

Given that Fe is acquired through the root and localized andutilized in all cells and various cellular compartments, regulatoryresponses must be coordinated for both environmental availabilityand metabolic demands. Identification of a master Fe sensor anddetermining how this sensor triggers signaling and transcriptionalregulatory networks that lead to proteomic and metabolomicsresponses is critical for developing a greater understanding of Fehomeostasis.

4.1. Building the Fe deficiency transcriptional regulatory network

For non-graminaceous plants, specifically, Arabidopsis, com-parisons to existing Fe-binding sensor proteins in bacteria andmammals hint at the possibility of putative E3 ligase BRUTUS (BTS)fulfilling the role of an Fe sensor, although further studies are nec-essary [3]. BTS is upregulated under Fe deficiency and knockoutmutants are embryo lethal [3,53], indicating that this protein mayhave critical functions in Fe transport to seeds or in Fe homeo-stasis during embryo development. However, bts-1 knockdownmutants exhibit increased tolerance of Fe deprivation [3]. Basedon yeast-2-hybrid assays, BTS interacts with bHLH proteins closelyrelated to POPEYE, a transcriptional regulator that appears to reg-ulate (directly and indirectly) a number of known Fe homeostasisgenes. Notably, pye-1 mutants have decreased tolerance to Fe depri-vation, and altered root and shoot Fe concentrations compared towild type [3]. Since BTS encodes a protein with several potentialFe binding sites, it is tempting to speculate that BTS affects Fehomeostasis via interaction with bHLH proteins related to PYE aftersensing Fe. In rice, the transcription factor IDEF1 (IDE-BINDINGFACTOR 1) regulates Fe homeostasis genes and also binds divalentcations such as Fe [54]. Notably, transcriptional activation does notappear to act through metal binding, and IDEF1 is not transcrip-tionally responsive to Fe deficiency. IDEF1 regulates expression ofIRO2, a bHLH transcription factor that is expressed in response toFe deficiency, which, in turn, positively regulates the Fe deficiencyresponse [54]. IDEF1 may also regulate the most recently identifiedbHLH component of the Fe regulatory network in rice, OsbHLH133.OsbHLH133 is transcriptionally induced under Fe deficiency, andrepresses translocation of Fe from root to shoot [5].

In most non-graminaceous plants the bHLH transcriptionalregulator, FER-Like Iron Deficiency Induced Transcription Factor(FIT) is one of the most important regulators of the Fe deficiencyresponse. In response to Fe deprivation FIT is produced and formsheterodimers with related bHLH proteins, bHLH38 and bHLH39.These heterodimers bind to and activate the expression of FerricReduction Oxidase 2 (FRO2), thus inducing reduction of Fe3+ toFe2+ and transport of Fe2+ from the rhizosphere into Arabidopsisroot epidermal cells through Iron-Regulated Transporter 1 (IRT1)[55–57]. For this reason FIT is thought to play an essential rolein facilitating Fe uptake from the rhizosphere in response to Fedeprivation.

Recently, researchers took advantage of a glucocorticoid (GR)inducible version of FIT (FIT-GR) to identify direct FIT targets. In thisstudy, researchers applied dexamethasone (a GR ligand) to induceFIT activity, followed by cycloheximide (a translational inhibitor),which enabled researchers to identify only FIT direct target genesby preventing protein synthesis of secondary targets [58]. Whileknown targets were identified, new targets such as MYB72, and


genes that are not FIT targets such as BTS and PYE were revealed.Using microarray analysis, this study also provided support forthe role of bHLH100/101 as a new and critical component of theFe deficiency regulatory network that regulates genes involved inrelocation of Fe independent of FIT [58].

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Predictive models for the activity of key Fe homeostasisranscriptional regulators such as FIT, PYE, bHLH38/39, andHLH100/101 from Arabidopsis and IRO2, bHLH133, and IDEF1rom rice can now be produced [12,14]. Additionally, the use ofhromatin immunoprecipitation followed by RNA-seq or microar-ay analysis (ChIP-seq or Chip-chip), Yeast-1-Hybrid screens,east-2-Hybrid screens, and other interaction studies will be help-

ul in reconstructing and connecting modules of the Fe deficiencyranscriptional regulatory network. These and other approachesan help identify transcriptional regulators of NRAMP3/4, YSL4/6,IT1, and other seed Fe homeostasis proteins. However, maintain-

ng the proper amount of biologically available Fe in the correctocation under Fe deficiency requires more than the activity ofensors and transcriptional regulators that control the availabil-ty of Fe chelators, reductases, and transporters. Additionally, theroduction of ROS scavengers, Fe storage proteins, Fe ligands,osttranscriptional and posttranslational modifications, and pro-ein degradation mechanisms are utilized to “tip the balance” andnsure that plants recognize when they have sufficient Fe and whenequestration is needed under conditions of excess Fe. Metabolicnd proteomic profiling are now helping to determine how theseesponses occur.

.2. Posttranscriptional and posttranslational response to Feeficiency

In a recent study, RNA-Seq and proteome analysis of Chlamy-omonas reinhardtii indicate that many transcripts associated withe assimilation are upregulated in response to Fe deficiencylong with their corresponding proteins [59]. This same studyhowed that transcripts and corresponding proteins that encodee-containing proteins are decreased, perhaps as part of an Fe spar-ng mechanism. Surprisingly, the change in abundance of around0% of the proteins analyzed did not correlate with the change inbundance at the transcript level. These findings suggested that, inome cases, low transcript abundance is due to a protein feedbackechanism, while in many other cases low Fe has a direct effect on

he biogenesis or integrity of Fe–sulfur proteins [59], thus, perhaps,ncreasing Fe availability for other metabolic processes.

Proteomic analysis of Arabidopsis with iTRAQ (Isobaric Tag forelative and Absolute Quantification) revealed that classic markersf the Fe deficiency response, including FRO2, NAS4, and a specu-ative plastidic Fe sensor involved in Fe-sulfur cluster assembly,re similarly regulated transcriptionally and post-transcriptionally60]. However, this same study shows that there is actually littleverlap between transcriptional and proteomics responses to Feeficiency, likely due to alternative splicing, RNA processing, andther posttranslational modifications [60]. In addition, new play-rs in Fe homeostasis were revealed in this study. One of theseroteins, eIF5a, is a conserved eukaryotic elongation factor, that

s not transcriptionally regulated by Fe deficiency, but may play aole in selective or arrested translation [61]. Additionally, a recentxciting study indicates that RNA polymerase II CTD-phosphatase-ike 1 (CPL1) plays a role in Fe deficiency response in addition toBA signaling [62]. Disruption of CPL1 causes upregulation of twoistinct clusters of genes, one enriched in ABA response genes, andnother in Fe homeostasis genes. In particular, cpl1 mutants exhibitncreased expression of IRT1, FIT, and FRO2 under Fe deficiency andncreased Fe in the root. cpl1 mutants also contain decreased Fen shoots under Fe sufficiency, yet increased tolerance to Fe defi-iency and cadmium. Since CPL1 is expressed strongly in the stele,



t does not directly regulate FIT (unless it is translocated to thetele). Rather, researchers propose that CPL1 acts upstream of FIT1nd the FIT regulon to keep Fe levels low in the root, most likely byacilitating xylem loading at the root. Sufficient Fe in the shoot thennhibits FIT-mediated epidermal uptake [62]. In addition to possible


changes in RNA synthesis, Fe deficiency responses are also medi-ated by microRNAs. A recent study indicates that since Fe deficiencyinhibits expression of Fe superoxide dismutase (SOD) genes, it actu-ally causes an increase in leaf copper concentration so that coppercan be utilized by copper SODs, which are functionally interchange-able with Fe SODs [63]. Notably, copper SOD transcripts increase inresponse to Fe deficiency, yet are degraded by miRNA398, which istranscriptionally repressed by Fe deficiency [63]. Further detailedstudies are needed to identify other RNA processing mechanismsinvolved in Fe homeostasis.

Using two complementary phosphoproteomics enrichmentmethods along with iTRAQ to detect global changes in phospho-proteins, researchers found that ribosomal phosphoproteins andphosphopeptides associated with RNA-binding proteins involvedin alternative splicing accumulate in response to Fe deficiency[64]. Ribosomal genes are also enriched in a transcriptional co-expression gene cluster that exhibits a bi-phasic expression patternwith rapid peaks in expression at 3 and 24 h under Fe deficiency[3], which are indicative of the importance of complex temporaland global changes in translation in responding to Fe deficiency.These findings are particularly striking as Perls/DAB, in combina-tion with DAPI staining, as well as Micro-PIXE reveal surprisinglyhigh concentrations of Fe specifically in the nucleus and nucleolusof pea embryos and leaf cells of Arabidopsis and tomato [26]. Thisis in contrast to animal cells, where nuclear localized Fe is associ-ated with disease [26]. Since the nucleolus is the site of ribosomalRNA biogenesis and high amounts of Fe are found in the nucleolus,it is possible that nuclear localized Fe may play a role in facilitat-ing ribosomal RNA biogenesis and translation [26]. Mutagenesisscreens with Micro-PIXE or Perls/DAB for genes in which nuclearlocalization of Fe is disrupted could help determine the function ofthis Fe pool.

Proteomic analysis with iTRAQ also revealed that the ubiquitinconjugating enzyme 13A (UBC13) [65], although not transcrip-tionally regulated by Fe deficiency, is also elevated in responseto Fe deficiency. Notably, it controls branched root hair forma-tion in response to Fe deficiency, suggestive of a role in mediatingdevelopmental responses. Yeast 2 hybrid analysis along with com-putational modeling indicate that UBC13 interacts with a numberof proteins, including a ubiquitin conjugating enzyme variant andan E3 ligase, to mediate the formation of Lys 63-linked ubiqui-tin chains on targets involved in root morphology and possiblyauxin transport [66]. Transcriptional profiling of the Arabidopsisroot in response to Fe deficiency also indicates that genes possiblyinvolved in protein ubiquitination, such as BTS and other ubiquitinligases and conjugating enzymes, are specifically enriched in a co-expression gene cluster that peaks at 48 h under Fe deficiency [3],suggesting that rapid and global increases in protein ubiquitinationplay a key role in the Fe deficiency response.

The biological relevance of protein ubiquitination has beenhighlighted in a recent study of the major Fe epidermal trans-porter, IRT1. This transporter, which is transcriptionally inducedin response to Fe deficiency, appears to undergo continuousmonoubiquitin-dependent cycling and turnover between theplasma membrane and early endosomes [67]. This process main-tains low IRT1 at the plasma membrane, ensuring that sufficient,but not excess Fe is taken up into epidermal cells. Additionally,cycloheximide treatments of plants exposed to Fe deficiency led tothe identification of a suite of genes that were induced under Fedeficiency by a rapidly turned over protein [58]. In this same studyIRT1 expression was elevated after cycloheximide treatments sug-


gesting that it is not only regulated by FIT but is also regulated bya transcriptional repressor that is rapidly turned over [58] (Fig. 1).Additionally, FIT itself is thought to be “active” (possibly throughposttranslational modification) under Fe deficiency, and stabi-lized by physical interaction with ethylene–induced transcription

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actors Ethylene Insensitive3 (EIN3) and Ethylene Insensitive3-ike1 (EIL1) [68,69]. However, FIT is subject to degradation, likelypon ubiquitination, when in an “inactive” state [68]. Finally, FRO2lso appears to be a target for posttranscriptional regulation, sinceverexpression of FRO2 causes a constitutive increase in tran-cript and an increase in reductase activity is only seen under Feeficiency [70]. Thus, we now have a more comprehensive under-tanding of how transcription control, ubiquitination, membraneocalization, and vesicular trafficking act synergistically to controle uptake in epidermal cells (Fig. 1).

Using two complementary phosphoproteomic enrichmentethods along with iTRAQ to detect global changes in phospho-

roteins, specific, and sometimes unique, Fe deficient-responsivehosphopeptides were identified within 425 distinct proteins [64].wenty-one phosphorylation motifs, which could potentially acts Fe deficiency signaling molecules in roots, were also described.urprisingly, plasma membrane H+-ATPases, which had previouslyeen shown to be activated by phosphorylation [71], were notmong the phosphoproteins found to be increased in the study.his finding is particularly striking considering that before FRO2nd IRT1 can reduce and transport Fe into the root epidermis inesponse to Fe deprivation, respectively, H+-ATPases, must firstacilitate acidification of the rhizosphere, increasing Fe solubil-ty [72]. Thus, further advances in methods that accurately detectlobal posttranslational modifications are needed.

.3. Metabolic responses to Fe deficiency: Shedding new light one ligands and respiration responses

Using the iTRAQ system to identify changes in the proteome, aumber of proteins involved in the methionine cycle appear to be

ncreased under Fe deficiency [60]. S-adenosyl methionine (SAM), product of the methionine cycle, is a precursor for the Fe lig-nd, NA. NAS, an NA synthase enzyme, which is transcriptionallyctivated by Fe deficiency [73], synthesizes NA from SAM. Recentetabolic profiling of sugar beet also indicates that increases in

oot NA, among other substances, occur in response to Fe fluctua-ions [74]. Additionally, energy-dispersive X-ray analysis detectedncreased insoluble Fe inclusion particles in specific organelles ofomato chloronerva NA-free mutants [75]. These studies show thathe synthesis of NA is regulated at multiple levels to properly con-rol the availability of soluble Fe (Fig. 1).

Proteins associated with the production of phenolic compoundsre also upregulated under Fe deficiency [60]. Phenolic compoundsnd organic acids are commonly reported in root exudates inesponse to Fe deficiency, presumably to chelate ferric Fe for sub-equent uptake from the rhizosphere [76]. In a more recent studyo determine whether such mechanisms are conserved betweenpecies, researchers confirmed that the production and secretionf phenolics and flavins from Arabidopsis and Medicago truncatula,espectively, is critical for Fe uptake under Fe deficiency [77].

Metabolic profiling of xylem sap and leaf extract of tomato,ugar beet, lupine, and peach tree exposed to Fe deficiency revealedncreases in TCA cycle metabolites, specifically organic acids that

ay play a role in Fe chelation [9]. In red clover these pheno-ic compounds chelate apoplastic Fe from cell walls and facilitateeutilization of Fe, independent of rhizosphere acidification [78].n addition, phenolic effluxers play a critical role in reutilization ofpoplastic Fe from rice stele cell walls [79]. Thus, metabolic profil-ng indicates that the synthesis of Fe ligands increases in response


o Fe deprivation to facilitate the translocation of Fe from differentources. Further use of such methods on Fe homeostasis mutantsuch as frd3, fpn1 and ysl1/ysl3 could indicate on how the interplayetween NA and citrate affects Fe localization and bioavailabilityhroughout growth and development.


Global transcriptional profiling of whole Arabidopsis rootsexposed to Fe deficiency for three days revealed increased expres-sion of genes involved in mitochondrial electron transport, inaddition to the TCA cycle. Researchers also reported increasedexpression of genes involved in anaerobic respiration underFe deficiency [80,81]. Phosphoproteomic profiling reveals thatphosphorylation of key carbohydrate metabolism enzymes, includ-ing phosphoenolpyruvate carboxylase and glucose-6-phosphatedehydrogenase, and altered accumulation of phosphopeptidesassociated with invertase, provide a molecular mechanism forincreased glycolysis under Fe deficiency [64]. Likewise, metabolicprofiling of sugar beet in response to Fe deficiency and Fe resupplyreveals increases in proteins associated with glycolysis, the TCAcycle, and anaerobic respiration, along with increases in organicacids, sugars, as well as NA [74]. Since Fe is a critical componentof many mitochondrial electron transport proteins, these find-ings suggest that plant roots respond to Fe deficiency stress withincreased fermentation rates, which provide sufficient NADH tocomplete oxidative phosphorylation [80]. Additionally, researchersconcluded from the study of sugar beet that anaplerotic reactionsfix carbon in the root, which is transported to the shoot [82,83]. Amore recent metabolic study of the xylem sap and leaf extract frompeach tree, lupine and tomato, and sugar beet indicated that notonly do these anaplerotic reactions take place in roots, but shootsas well [9]. While xylem sap contains decreased levels of aminoacids and carbohydrates, leaf extracts contain more [9], suggest-ing that anaplerotic reactions, using amino acids as carbon sources,occur in Fe-deficient leaves also to maintain respiration.

These metabolic studies are particularly exciting from a systemsbiology perspective. Flux analysis of enzymatic activity and themetabolites produced from enzymes involved in metabolic pro-cesses can be used to create mathematical models that describehow enzymatic processes change under conditions of fluctuatingFe availability. Such models could also include transcriptional reg-ulatory components, signaling response, such as phosphorylationstatus, and even Fe movement rates, similar to that seen with auxinflux in roots [84].

Interestingly, many metabolic changes appear to be reflective ofcell-specific response to Fe deprivation revealed by cell-specific andgenome-wide transcriptional profiling. Although there is a core setof genes that are impervious to stress response, similar cell-specifictranscript profiling under salt stress indicated that a number genes,particularly those involved in carbohydrate metabolism, respondto different stress conditions in a cell specific manner [2]. Forexample, compared to other cell types, the cortex transcriptomeis enriched in genes associated with photosynthesis, regardless ofenvironmental conditions, and chloroplasts accumulate in light-grown cortex cells [2]. However, in Fe-deprived root cortex cellsthere is an increase in expression of these same genes yet deceasedexpression of carbohydrate transmembrane transporters, perhapsto inhibit carbohydrate translocation from the shoot to the root[2,3]. Compared to other cell types, the endodermis exhibits strik-ing decreases in the expression of genes encoding fructokinase,glucokinase, and hexokinases in response to Fe deficiency, whichmay play a role in carbohydrate metabolism as well. These findingssuggest that although Fe is primarily detected in the vasculature inroots, as it traverses from the epidermis to the vasculature someof it is captured, utilized, and stored within cells internal to theepidermis such as the cortex and endodermis for metabolism. Fetransporter Ferroportin 2 (FPN2) is thought to move Fe from thecytoplasm into the vacuole in cortex and epidermal cells under Fe


deficiency, presumably in order to buffer excess Fe intake [40], butit may also play a role in metabolic requirements specific to theendodermis and cortex. Metabolic and proteomic profiles are nowavailable for specific root cells under nutrient sufficient conditions[85,86]. Querying and comparing data from these studies to similar

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ata obtained under Fe deficiency could allow researchers to recon-truct high-resolution transcription and metabolic maps of the Feeficiency response.

. Concluding remarks and future perspectives

Today, large-scale datasets have been produced and can besed in combination to begin to make testable and predictiveegulatory models of Fe homeostasis [12,13]. The most recent mod-ling efforts consist mainly of co-expression networks, which onlyllow researchers to predict regulatory relationships between tran-cription factors and their targets, and provide candidate genesnvolved in these networks. Exploration of small and large-scalerotein–DNA and protein–protein interaction data should increasehe strength of correlations between interactors identified in co-xpression networks. Proteome and metabolome profiling datarovide an even richer source of information about signaling andther posttranscriptional processes involved.

Moreover, recent advances in localization methods for Fe andther metals now enable researchers to link large-scale data tohanges in Fe translocation. While the most desirable detectionools would enable in vivo detection and measurement of Fe andther metals, such tools are currently lacking in the field of plantiology. Instead, histochemical methods and advanced ionic quan-ification techniques have led to the creation of high-resolutionlemental maps at specific time points, which enable visualizationnd measurement of Fe and other metals at cellular and subcellularesolution. While there are advantages and disadvantages to each ofhese methods, continued advances that increase resolution, sen-itivity, and usability are sure to contribute to rapid advances inhe field. Indeed the use of such methods has already increasedur understanding of where Fe is localized in plants and whatransporters and ligands contribute to this localization (Table 1).tilizing techniques such as synchrotron X-ray fluorescence micro-

omography or micro-PIXE on known regulatory, signaling, andetabolic mutants in combination with large-scale datasets, or for

creening mutants of genes derived from large-scale data, can helpuild more inclusive, dynamic Fe homeostasis models. Such mod-ls could then include mathematical descriptors of how changesn Fe availability affect Fe translocation and the many biologicalrocesses that require Fe.

There are a number of caveats to this approach that should beept in mind, one being that the identification of network nodesan be confounded by redundancy, making study of single mutantsutile. This could possibly be alleviated with miRNA approaches thatarget gene families. Also, more accurate predictive models requirepatial resolution due to the distinct identity of cell types. Finally,etabolic flux data, which would likely provide the most predic-

ive power for generating models for Fe homeostasis, is still quitehallenging to obtain in the area of plant biology [1,87]. However,nclusion of metabolic rates for key enzymes such as H+ ATPases,e reductases, and phosphoenolpyruvate carboxylase could lead tohe reconstruction of testable regulatory and metabolic network

odels that couple Fe uptake, translocation, and assimilation toetabolic processes such as photosynthesis and respiration, and,

ltimately, growth.Indeed, the long-term goals of many research efforts in this field

re increased growth and nutrient content of agriculturally impor-ant crops. These goals are particularly urgent due to predictedncreases in global food demand in the coming years. The increas-



ng use of non-model species indicated in many studies above is huge step toward those goals. Additionally, although other met-ls are not featured in this review, many studies listed above doot consider Fe in isolation. This is critical, given that Fe bioavail-bility affects and is affected by homeostasis of other metals and

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metabolic processes. Perhaps then, the use of systems biology isparticularly appropriate, as it will enable us to maintain a globalperspective of the side effects produced through genetic manip-ulation of Fe homeostasis. Such a perspective, along with the useof high-resolution imaging tools, makes for exciting times as wesearch for ways to affect Fe bioavailability in a sustainable way.

Acknowledgments

We thank Dr. Devarshi Selote, Dr. Jeff Gillikin, Dr. RosangelaSozzani for critical review of the manuscript.

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