REDUCTION AND UPTAKE OF ffiON IN PLANTS

ERIN L. CONNOLLY and MARY LOU GUERINOT

Department of Biological Sciences
6044 Gilman, Dartmouth College
Hanover, NH 03755

1. Introduction

Iron, an essential nutrient, is not readily available to plants growing in the soil.
Although iron is the fourth most abundant element in the earth's crust, it is
found mainly as stable, insoluble oxyhydroxide polymers that effectively limit
free Fe(III) to an equilibrium concentration of 10" 17 M at neutral pH, a value far
below that required for the optimal growth of plants. In addition to the problem
of iron's solubility, the chemical properties of iron require cells to place
limitations on its accumulation. Fe(II) and Fe(III) can act catalytically to
generate hydroxyl radicals that are able to damage cellular constituents such as
DNA and lipids (Halliwell and Gutteridge, 1992). Thus, the uptake of iron is a
highly regulated process, as cells balance meeting their demands for this
essential element against its potential for wreaking cellular havoc.
Plants use a variety of strategies to dissolve Fe(III) oxides: protonation,
reduction and chelation. Protonation (defined here to mean release of protons)
drives more Fe(III) into solution by shifting the equilibrium in favor of
dissociation of the Fe(OH)3 complexes; the solubility of Fe(III) increases 1000-
fold with each one unit decrease in pH. Chelation largely involves the release of
small molecular weight, high-affinity Fe(III) chelators synthesized and secreted
under conditions of iron limitation. Once chelated, the Fe(III) remains in
solution. Reduction of Fe(III) to Fe(II) relies on membrane bound Fe(III)
reductases and offers the benefit that Fe(II) is much more soluble in the
rhizosphere than Fe(III) at a given pH. For comparison, Fe(II) is approximately
10 16-fold more soluble than Fe(III) at neutral pH (Neilands, 1991 ).
The three means of solubilizing iron described above are often assigned to
two main Strategies (Figure 1): Strategy I which relies on protonation and
reduction and Strategy II which relies on chelation (for reviews see Guerinot
and Yi, 1994; Mori, 1998). Because this chapter focuses on recent developments
in our understanding of the Strategy I response in dicotyledonous plants, we will
first provide a brief summary of Strategy II.
179
H. Asard et al. (eds.), Plasma Membrane Redox Systems and their Role in Biological Stress and Disease
© Springer Science+Business Media Dordrecht 1998
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Grasses, including barley, wheat, rye, maize and rice, synthesize and release
Fe(III)-chelating compounds called phytosiderophores in response to iron-
deficiency. The Fe(III)-phytosiderophore chelates are then taken up by the plant
via a yet to be identified transport system. The phytosiderophores that have been
characterized to date are all structurally related and have been generically
termed mugeneic acids after the first phytosiderophore that was characterized.
This Strategy is similar to that employed by many bacterial and fungal species
(Guerinot, 1994).
All remaining plant species are thought to rely on a reductive mechanism to
solubilize rhizosphere Fe(III) (Guerinot and Yi, 1994). This Strategy, termed
Strategy I (Marschner et a/., 1986), is thought to be an obligatory step in iron
uptake (Chaney et a/., 1972). The initial reduction of Fe(III) is carried out by a
plasma membrane-bound Fe(III) chelate reductase (Buckhout et a/., 1989). The
resulting Fe(II) is then transported across the root epidermal cell membrane by a
Fe(II)-specific transporter (Eide et a!., 1996). Both the Fe(III) chelate reductase
(Bienfait et a!., 1989; Yi and Guerinot, 1996) and the Fe(II) transport activities
(Fox et a/., 1996) are enhanced under iron-deficiency. In addition, proton
secretion is enhanced under iron-deficiency (Bienfait, 1985) and serves to
acidify the rhizosphere. In some species, the Strategy I response also includes
morphological changes such as the development of root hairs and transfer cells
as well as the excretion of phenolic compounds and flavins (Marschner eta!.,
1986).

Strategy I Strategy II

y
(Arnbidopsis, pea, tomato) (barley, maize, oat)
ATP
Siderophore
Soil
DP Particle
Ferric
ADH Fe( II I)-chelate Siderophore

Fe(ll) ~ 1 ~ ~ ..,,,,,,
Fe(ll) . , . . / Degradation
IRTI

in ide outside in ide outside

p I p I

Figure 1. Models for iron acquisition by higher plants. Only the best studied features are
shown (modified from Guerinot and Yi, 1994).
 181

The Strategy I response of higher plants is very similar to the iron uptake
mechanism in the baker's yeast, Saccharomyces cerevisiae (for reviews see
Eide, 1997; Eide and Guerinot, 1997; Askwith and Kaplan, 1998); indeed, the
availability of yeast iron uptake mutants has proven invaluable in the study of
iron uptake in plants. A similar iron uptake mechanism is also thought to be
involved in iron uptake in the unicellular green algae, Chlamydomonas
reinhardtii (Eckhardt and Buckhout, in press; Lynnes et a/., 1998), and
Chlorococcum littorale (Sasaki eta/., 1998).
In this chapter, we will review progress to date on identification of the genes
involved in iron uptake in Strategy I plants. One theme that we will emphasize is
the power of a multi-organism approach. For example, the ability to utilize
information generated from genomic sequencing efforts in various organisms
and the ability to use yeast as a model has greatly enhanced our understanding
of metal uptake in plants. We will also include information here on plant
mutants that are impaired in iron uptake as these mutants are a valuable resource
for furthering our understanding of iron uptake in plants.

2. Fe(III) Reduction

In response to iron deprivation, Strategy I plants up-regulate root Fe(III) chelate

reductase activity, which oxidizes NAD(P)H and reduces Fe(III) to Fe(II) at the
root surface; the membrane-bound enzyme transfers electrons from the cytosol
to the external electron acceptor- Fe(III) chelates. Under normal, aerobic soil
conditions, the reduction of Fe(III) to Fe(II), rather than the transport of Fe(II),
is thought to be the overall rate-limiting step in iron uptake (Grusak et a/.,
1990). Once inside the plant, Fe(II) is oxidized to Fe(III) and transported
through the xylem as Fe(III)-citrate (Tiffin, 1970). It is thought that a Fe(III)
chelate reductase must be present in the aerial portions of the plant in order to
reduce Fe(III) prior to transport into the leaf cells. Indeed, Fe(III) reductase
activity has been detected in leaves of sunflower (de Ia Guardia and Alcantara,
1996) and Vigna unguiculata (Brliggemann et a/., 1993 ). Fe(III) chelate
reductase activity also appears to be developmentally regulated, with Fe(III)
reductase activity increasing during seed fill to supply the developing seeds
(Grusak, 1995).
Bienfait (Bienfait, 1985; Bienfait et a/., 1983) proposed that root epidermal
cells express two distinct Fe(III) chelate reductase activities. One activity,
termed the "standard" reductase, is thought to be capable of reducing
ferricyanide and is constitutively expressed. The second activity, termed the
"turbo" Fe(III) chelate reductase activity, is induced in response to iron
deprivation. This inducible Fe(III) reductase activity is thought to be capable of
reducing both Fe(III) chelates and ferricyanide. Recent work addressing this
hypothesis is somewhat contradictory. Studies of the NADH-dependent
182

reduction of Fe(III) chelates by purified plasma membranes isolated from

various Strategy I plants have failed to detect a iron-deficiency inducible Fe(III)
chelate reductase activity that is biochemically distinct from the constitutive
Fe(III) chelate reductase activity that is detected in iron-sufficient plants
(Buckhout et al., 1989; Holden et a!., 1991; Moog and Briiggemann, 1995;
Rubinstein and Luster, 1993 ). These studies supported the hypothesis that
Fe(III) chelate reductase is either activated or synthesized in increased amounts
in response to iron-deficiency. A recent study examined the pH dependence of
the "turbo" Fe(III) chelate reductase versus the constitutive Fe(III) chelate
reductase both in vivo and in purified plasma membrane from Beta vulgaris
(Susin et a/., 1996). The study demonstrated that, at low pH, the turbo reductase
activity is distinct from the constitutive reductase activity and thus lent support
to the idea of a novel inducible Fe(III) chelate reductase. Further studies are
required to definitively answer this question.
Recent progress has been made towards the biochemical purification of
Fe(III) reductase. Partial purification of the Fe(III) chelate reductase from
tomato roots implicated a 35-kDa polypeptide as a potential component of the
reductase (Holden eta/., 1994). A 200-kDa, NADH-dependent Fe(III) chelate
reductase protein complex from plasma membrane preparations of tomato root
has recently been isolated (Bagnaresi et a/., 1997). Protein complexes of 200
kDa were also found in microsomal fractions. Subsequent purification of the
200 kDa complexes allowed the resolution of a 28 kDa subunit which is thought
to correspond to a 32 kDa polypeptide as separated by SDS-PAGE. The purified
Fe(III) chelate reductase protein complex resembled that isolated from maize
(Bagnaresi and Pupilla, 1995), suggesting that Strategy I and Strategy II plants
have similar Fe(III) reductases that appear to belong to a family of cytochrome
b5 reductase flavoproteins (Bagnaresi et al., 1997; Berczi et a!., 1998). This
result is consistent with what we know about the previously cloned Fre 1 (Dancis
eta/., 1992) and Frp1 (Roman eta!., 1993) proteins, which are thought to form
a family of plasma membrane electron transport proteins responsible for
mobilizing cytoplasmic reducing equivalents and donating them to extracellular
substrates (Roman eta/., 1993; see section 3 below).

3. Genes Involved in Iron Reduction in Strategy I Plants

Genes encoding Fe(III) chelate reductases have now been cloned from plants
using two different approaches. Using degenerate PCR with primers
corresponding to conserved regions in the flavin- and NADPH-binding domains
of the S. cerevisiae and S. pombe Fe(III) reductase proteins, several candidate
genes (froh for ferric reductase/Qxidase homologue) that may encode Fe(III)
reductases have been identified in Arabidopsis (Figure 1) (Robinson et a/.,
1997; Sadjuga et a/., 1997). One of these genes, .frohB, maps to the same
 183

location as the frdl (ferric reductase gefective) mutation, which had previously
been shown to result in a lack of inducible root Fe(III) chelate reductase activity
in Arabidopsis (Yi and Guerinot, 1996). Indeed, we have now shown that lines
carrying various alleles of frdl have mutations in frohB and we have
successfully complemented the frdl mutant with a genomic clone of frohB
(Robinson et al., in preparation). We also know that jrohB is more highly
expressed in iron deficient roots than in iron sufficient roots.
The other successful approach to cloning a Fe(III) chelate reductase gene
from a plant species utilized reverse genetics. Microsequence data from purified
Fe(III) reductase isolated from maize root microsomes allowed the identification
of a maize eDNA (NFR for NADH-dependent-ferric reductase) that is predicted
to encode a cytochrome b 5 reductase (J-F Briat, personal communication). This
reductase is likely to reside on the ER and is not predicted to be involved in
uptake ofF e(III) from the rhizosphere.
The froh genes and the maize gene all encode b-type cytochromes belonging
to a larger family whose other members include the respiratory burst oxidase of
mammalian neutrophils (gp91phox). The neutrophil oxidative burst occurs in
response to pathogen challenge and results in the production of active oxygen in
a reaction that is catalyzed by a plasma membrane NADPH oxidase (Chanock et
al., 1994; Henderson and Chappell, 1996). The oxidase is composed of gp91phox
and p22phox, two plasma membrane proteins which together form cytochrome
b558 (Parkos et al., 1987; Segal et al., 1998). Recently, additional homologs of
gp91phox have been isolated from rice [rbohA for respiratory hurst Qxidase
homologue (Groom et al., 1996)] and Arabidopsis [rbohA (Keller et al., 1998;
Murphy et al., 1998)]. These plant homo logs show extensive sequence similarity
to gp91phox at their C-termini. Interestingly, the predicted proteins differ from
gp91phox in that they contain large hydrophilic N-terminal domains with potential
Ca2+-binding EF hand motifs and have similarity to a human GTPase activating
protein (Keller et al., 1998). All of the reductases are involved in passing one
electron across a membrane to various acceptors, Fe(III) in the case of the iron
reductases and molecular oxygen in the case of the respiratory burst oxidases
(for a review of NAD(P)H-utilizing oxidoreductases of the plasma membrane
see (Berczi and Asard, 1995; Berczi et al., 1998).

4. Plant Mutants with altered Iron Uptake Activities

In this section, we will briefly describe several of the iron uptake mutants of
Arabidopsis, tomato and pea (Table 1). When the complete sequence of the
Arabidopsis genome becomes available, we will be in an excellent position to
identify the genes that are affected in each of the mutants, provided the
mutations have been well mapped. Cloning tomato genes identified ·by well
184

mapped mutations is also possible. Unfortunately, the large size of the pea
genome is hampering map-based cloning efforts in this species.

4.1. frdMUTANTS OF ARABIDOPSIS

Arabidopsis mutants,frdl andfrd3, that exhibit defects in Fe(III) reduction have

been identified (Yi, 1995; Yi and Guerinot, 1996). frdl mutants do not show
induction of Fe(III) chelate reductase activity under iron-deficiency; however,
these mutants are still capable of acidifying the rhizosphere in response to iron
limitation. Furthermore, frdl mutants have lost the ability to reduce Cu(II)
chelates. These mutants also do not translocate radiolabeled iron into the shoots
when roots are presented with a tightly chelated form of Fe(lll). This confirms
that iron must be reduced before it can be transported and that Fe(lll) reduction
can be separated from proton secretion.
frd3 mutants express Fe(III) reductase activity under both iron-deficient and
iron-sufficient conditions. They also show constitutive expression of the iron-
regulated metal transporter, IRTJ (Eide et ai., 1996). Moreover, frd3 mutants
exhibit proton release under both iron-sufficient and iron-deficient conditions
(Fox and Guerinot, unpublished data). Taken together, these data suggest that
frd3 mutants may be either regulatory or transport mutants. Efforts to clone the
gene identified by thefrd3 mutation are now underway.

4.2. man MUTANT OF ARABIDOPSIS

Using a screening procedure based on X-ray fluorescence spectrometry

(Delhaize et ai., 1993), an EMS mutant of Arabidopsis was identified that
accumulated manganese in its leaves. This mutant, called man] (for manganese
accumulator), actually accumulates a range of metals (Delhaize, 1996). man]
seedlings accumulate Mn, Cu, Zn and Mg in leaves when grown either in soil
or hydroponically. The roots of man] seedlings also accumulate metals but
unlike leaves, they accumulated I 0-fold more iron than wild type roots. This
accumulation may be explained by that fact that the man] mutant plants exhibit
an increase in root ferric chelate reductase activity relative to wild type. Thus,
the man] mutation may affect a regulatory gene that is involved in the control of
metal uptake or metal ion homeostasis (Delhaize, 1996). The mutation has been
mapped to the upper arm of chromosome 3 and is linked to the phenotypic
marker hy-2.

4.3. chin MUTANT OF TOMATO

The chioronerva (chin) mutant of tomato accumulates iron (Stephan and Scholz,
1993). Chin is a recessive mutation that affects a gene involved in the synthesis
of the non-protein amino acid nicotianamine (NA); application of NA to the
 185

leaves or roots of mutant plants rescues the mutant phenotype (Stephan and
Scholz, 1993). Chin phenotypically resembles an iron-deficient plant despite
high iron levels in the leaves. It has been proposed that NA is somehow
involved in regulation of iron-deficiency responses in Strategy I plants through
its ability to selectively complex with Fe(II) (Pich et al., 1991 ). Because the
mutant has no NA, it suffers from apparent iron-deficiency and fails to down-
regulate inducible iron-uptake processes. In addition, NA may be involved in
mediating iron transport in phloem (Stephan and Scholz, 1993 ).

TABLE I. Iron uptake mutants in Strategy I plants

Mutant Defect Species Phenotype Reference

frdl Fe(III) chelate Arabidopsis Chlorotic Yi and Guerinot, 1996

reduction

frd3 Constitutive Fe(III) Arabidopsis Chlorotic Yi, 1995

reduction

man] Overaccumulates Arabidopsis Chlorotic Delhaize, 1996

various metals

chin Nicotianamine Tomato Chlorotic Ling et a!., 1996

synthesis Retarded growth

fer Fe(III) chelate Tomato Chlorotic Brown eta!., 1971

reduction and
acidification

brz Overaccumulation Pea Necrotic spots Welch and Kochian,

of iron on leaves 1992

dgl Overaccumulation Pea Necrotic spots Gottschalk, 1987

of iron on leaves Kneen et a/., 1990

A recent study examined alterations in protein patterns in response to iron

limitation in wild type and chin tomato plants. Herbik et al. (1996) found that
three polypeptides corresponding to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), formate dehydrogenase (FDH) and ascorbate peroxidase (AP)
showed increased expression in the roots of iron-deficient wild type plants as
well as in the roots of both iron-deficient and iron-sufficient chin plants relative
to iron-sufficient wild type plants (Herbik eta!., 1996). The authors propose that
these three enzymes may be involved in iron-deficiency stress responses. Chin
mutants exhibit copper-deficiency in leaves but not roots presumably because
186

NA is required to transport copper via the xylem to the leaves (Pich et ai.,
1994). Copper-deficiency in chin leaves was shown to lead to dramatic
reductions in chloroplastic and cytosolic Cu-Zn superoxide dismutases and
plastocyanin (Herbik et ai., 1996). The chin mutation has been mapped onto the
high density RFLP map of tomato in anticipation of walking to the gene (Ling et
ai., 1996).

4.4. fer MUTANT OF TOMATO

The fer (Ee ~fficiency reactions) mutant of tomato is chlorotic and is unable to
respond to iron-deficiency (Brown et ai., 1971 ); the mutant lacks the ability to
switch on Strategy I responses including proton release and Fe(III) chelate
reductase activity. These data suggest that FER may encode a positive regulator
of proteins like Fe(III) chelate reductase (Bienfait, 1988). A recent study has
shown that fer is epistatic to chin (Ling et ai., 1996), supporting a role for Fer in
regulating chin. The fer mutation has been mapped onto the high density RFLP
map of tomato and a chromosome walk has been initiated to isolate the fer gene
by map-based cloning (Ling et ai., 1996).

4.5. brz MUTANT OF PEA

The brz (bron?;e) mutant of pea, E 107, accumulates 50 times more iron in its
leaves than does wild type and also develops bronze necrotic spots on its leaves
(Kneen et ai., 1990; Welch and Kochian, 1992). The basis for excess iron
accumulation appears to be increased rates of Fe(III) reduction; brz plants had
high rates of Fe(III) reduction regardless of plant iron status. Iron transport
activity is unaltered in the brz mutant. brz mutant plants also accumulate high
levels of Mg(II), Mn(II) and Zn(II) which is consistent with a role for Fe(III)
reductase activity in general cation uptake (Welch et ai., 1993 ).

4.6. dgi MUTANT OF PEA

The phenotype of the dgi (.Qegenerative leaves) mutant of pea (Gottschalk,

1987) is very similar to that of the brz mutant although the two mutations are
not allelic. dgi mutant plants develop necrotic spots on the leaves and
accumulate iron in vegetative tissues (Kneen et al., 1990). A recent study
examined Fe(III) reductase activity in wild type and dgi plants with reciprocal
shoot: root grafts. The results showed that the dgi mutation allows the expression
of a shoot factor that leads to an increase in Fe(III) chelate reductase activity in
roots (Grusak and Pezeshgi, 1996). Taken together, these results suggest that the
brz and dgl mutations may affect the regulation of ferric reductase activity.
 187

5. Iron Transport

Iron, like other metal ions, needs to be transported from the soil solution into the
root and then distributed throughout the plant, crossing both cellular and
organellar membranes. Transport across the plant plasma membrane is driven by
an electrochemical gradient of protons generated by plasma membrane
H+-ATPases (Briskin and Hanson, 1992). These primary transporters pump
protons out of the cell, thereby creating pH and electrical potential differences
across the plasma membrane. Secondary transport systems then utilize these
gradients for many functions, including nutrient uptake. A number of genes
involved in metal ion transport in plants have already been identified on the
basis of function, via complementation of yeast mutants, or on the basis of
sequence similarity. Not surprisingly, many of these genes belong to previously
described transporter families including those encoding P-type ATPases
(Axelsen and Palmgren, 1997) and Nramp proteins (Cellier et al., 1995).
An Arabidopsis gene encoding a presumptive Fe(II) transporter, IRTJ (for
Iron Regulated Transporter), was isolated because its expression in yeast could
restore iron-limited growth to a yeast fet3fet4 mutant defective in iron uptake
(Eide et al., 1996). Consistent with its proposed role as a metal ion transporter,
yeast expressing IRTJ possess a novel iron uptake system that is specific for
Fe(ll) over Fe(lll). Fe(II) uptake was not greatly inhibited by high
concentrations of other physiologically relevant metal ions such as Cu(I), Cu(II),
Mn(II), and Zn(II). Most interestingly, cadmium has been shown to inhibit
Fe(II) uptake by IRTI. This suggests that cadmium may serve as a substrate for
this transporter. In Arabidopsis, IRTJ is expressed in roots and is induced by
iron-deficient growth conditions. Based on these results, we proposed that IRTI
is an Fe(II) transporter that takes up iron from the soil. Interestingly, IRTl bears
no resemblance to either the high affinity (Ftrl p; Stearman et al., 1996) or the
low affinity (Fet4p; Dix et al., 1994) iron transporters in yeast (Askwith and
Kaplan, 1998).
The significance of IRTI in the field of metal uptake research is two-fold.
IRTJ is the first iron transporter gene to be isolated from plants and it provides a
useful handle on the mechanism and regulation of iron uptake in plants. Second,
IRT I has led to the discovery of a new family of transporters involved in metal
ion uptake (Grotz et al., 1998). We have named this group the "ZIP" gene
family (for ZRT/IRT-related £roteins) for the first three members to be isolated
and characterized, ZRTl, ZRT2, and IRTI. ZRTJ and ZRT2 encode Zn
transporters in S. cerevisiae (Zhao and Eide, 1996a; 1996b). These ZIP proteins
define a family of metal ion transporters that are found in plants, protozoa,
fungi, invertebrates, and vertebrates, making it now possible to address
questions of metal ion accumulation and homeostasis in diverse organisms. To
date, six of the family members have been implicated in metal ion transport
(Eide et al., 1996; Zhao and Eide, 1996a; 1996b; Grotz et al., 1998). The ZIP
188

family is structurally distinct from other metal ion transporters such as the CDF
family (Paulsen and Saier, 1997) that includes the recently identified
mammalian zinc effluxers, P-type ATPases that are involved in the transport of
a variety of cations, and the Nramp proteins recently implicated in divalent
cation transport in mammals (Belouchi eta/., 1997; Fleming eta/., 1997).
With many cloned genes already in hand, the obvious challenge now is to
decipher the role of each of the transporters encoded by these genes. For most
transporters, physiologically important information such as expression pattern
and mechanism of regulation is still lacking. Various molecular approaches
ultimately can tell not only in what tissue and cell types certain transporters are
expressed but where within a cell each is expressed. They can also tell whether
gene expression is directly influenced by changes in cation concentrations. We
are also now in a position to identify plant mutants carrying insertions that
eliminate the function of particular transporter genes (Krysan et a/., 1996;
McKinney et a/., 1995); this will greatly help in assigning functions. Having
cloned genes is also allowing us to undertake structure-function studies on the
encoded proteins themselves. Many transporter activities have been well
characterized at the electrophysiological level. Our ability to now combine such
information with structural information about the proteins will hopefully lead to
an understanding of the molecular mechanisms of transport. Finally, moving
beyond how any one transporter functions, we need to keep in mind that
ultimately we want to understand cation transport at the whole plant level and to
use such knowledge to create plants that selectively accumulate or exclude
various metals.

6. Acknowledgements

We thank Elizabeth Rogers, Natasha Grotz and Rob McClung for critically
reading the manuscript. Work in the laboratory of M.L. Guerinot is supported
by grants from NSF (IBN-9643998) and DOE (07-97ER20292).

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