Journal of Experimental Botany, Vol. 57, No. 4, pp. 711–726, 2006
doi:10.1093/jxb/erj073 Advance Access publication 10 February, 2006
REVIEW ARTICLE
The significance of amino acids and amino acid-derived
molecules in plant responses and adaptation to heavy
metal stress
Shanti S. Sharma1 and Karl-Josef Dietz2,*
1
Department of Biosciences, Himachal Pradesh University, Shimla 171 005, India
Department of Physiology and Biochemistry of Plants, Faculty of Biology, University of Bielefeld,
D-33501 Bielefeld, Germany
2
Received 16 February 2005; Accepted 22 November 2005
Abstract
Plants exposed to heavy metals accumulate an array of
metabolites, some to high millimolar concentrations.
This review deals with N-containing metabolites frequently preferentially synthesized under heavy metal
stress such as Cd, Cu, Ni, and Zn. Special focus is
given to proline, but certain other amino acids and
oligopeptides, as well as betaine, polyamines, and nicotianamine are also addressed. Particularly for proline
a large body of data suggests significant beneficial
functions under metal stress. In general, the molecules
have three major functions, namely metal binding,
antioxidant defence, and signalling. Strong correlative
and mechanistic experimental evidence, including
work with transgenic plants and algae, has been provided that indicates the involvement of metal-induced
proline in metal stress defence. Histidine, other amino
acids and particularly phytochelatins and glutathione
play a role in metal binding, while polyamines function
as signalling molecules and antioxidants. Their accumulation needs to be considered as active response
and not as consequence of metabolic dys-regulation.
Key words: Adaptation, amino acids, heavy metal stress, Ncontaining metabolites, proline, stress defence.
Introduction
Uptake of excess metal ions is toxic to most plants. The
biochemical impact of metal ions on the cells is as diverse
as their chemical nature. From the approximately 90
elements present in the earth’s crust, about 80% are metals
and 60% are heavy metals with specific weights higher than
5 g cmÿ3. In context of metal toxicity, other elements with
only partial metal properties such as As and with a specific
weight lower than 5 g cmÿ3 such as aluminium also need to
be considered due to their toxicity to plants. In the literature, the term ‘heavy metal’ is occasionally used with a very
broad and misleading meaning. Based on metal coordination chemistry, Nieboer and Richardson (1980)
provided a biologically relevant classification of metals
into three categories namely, class A with affinity for
O-containing ligands, class B with affinity for N- or
S-containing ligands, and borderline, i.e. intermediate between the two with affinity for all three groups of ligands
with definite preferences. This categorization reflects
a general manner in which different metal ions interact
+
2+
3ÿ
with biological systems. Al3+, AsOÿ
2 , AsO4 , Au , Cd ,
+
2+
2+
3+
2+
3+
2+
2ÿ
Cu , Cu , Co , Cr , CrO4 , Fe , Fe , Hg , Mn2+,
2+
6+
2+
2ÿ
Ni2+, Pb2+, SeO2ÿ
are
3 , SeO4 , Sn , W , and Zn
examples of ions with known toxicity to plants. At present, most studies focus on a few metal ions such as Al, Cd,
Hg, Ni, and Zn. Typically, root inhibition is observed
at micromolar concentrations, i.e. at concentrations more
than three to four orders of magnitude lower than is usually
encountered in salinity stress.
Phytotoxicity of heavy metals in most parts can be
attributed to symplastic accumulation of heavy metals,
particularly in the plasmatic compartments of the cells, such
as the cytosol and chloroplast stroma (Brune et al., 1995).
Metal-induced changes in development are the result of
either a direct and immediate impairment of metabolism
(Woolhouse, 1983; Van Assche and Clijsters, 1990) or
signalling processes that initiate adaptive or toxicity responses that need to be considered as active processes of
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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712 Sharma and Dietz
the organism (Jonak et al., 2004). Transport processes
have been recognized as a central mechanism of metal
detoxification and tolerance (Hall, 2002; Hall and Williams,
2003). Some metals, for example, Zn and Cu, are essential
for normal plant growth and development as they serve as
structural and functional components of specific proteins.
Other metals, for example, Cd and Pb, have no known
function in plants although a Cd requirement for carbonic
anhydrase from marine diatoms has been reported (Lane
and Morel, 2000).
The generalized dose–response curves for the two kinds
of metals differ with regard to their effects on plant growth.
Whereas for non-essential metals these curves comprise
a no-effect and a toxicity zone, for essential metals the
response curves show an additional deficiency zone preceding the no-effect zone of adequate supply. It is implied
in both cases that the plants are endowed with an inherent
capability of tolerating toxic metals to some extent. Metal
ions turn toxic as soon as their concentration exceeds a
metal-specific threshold which varies strongly among plant
species and ecotypes, and also with metal properties. As an
exception to the rule, certain low concentrations of nonessential elements have occasionally been associated with
some promotion of plant growth, as for example seen for
Alyssum species and Thlaspi goesingense (Küpper et al.,
2001).
Populations of certain plant species that chronically
experience exposure to elevated metal concentrations, for
example, those inhabiting metal-enriched locations, have
repeatedly evolved tolerance to the metal(s) in question
(Ernst et al., 1990; Schat et al., 1996). Such metal-tolerant
ecotypes and genotypes are the examples of accelerated
(micro)evolution when the selection pressure is acute.
Examples of tolerant species are Arabidopsis halleri,
a Zn-hyperaccumulator, Thlaspi species, that are Cd-/Znor Ni-hyperaccumulators, Silene vulgaris with Zn-, Cu-,
and Cd-resistant ecotypes, and Alyssum bertolonii, a
Ni-hyperaccumulator (Ernst and Nelissen, 2000; Küpper
et al., 2001; Bert et al., 2003; Freeman et al., 2004). Correspondingly, the dose–response curves of metal-tolerant
variants exhibit a wider no-effect or even limited beneficial
zone when compared with their non-tolerant counterparts.
Apparently, while evolving tolerance, they acquired some
specific molecular means of efficiently detoxifying the
surplus toxic metal ions through a combination of complexation and safe deposition. Such acquisition of special
tolerance traits is not available to non-tolerant genotypes.
Metal hyperaccumulation is defined based on certain metalspecific thresholds of metal levels detected in the shoots.
Thus, plants with an ability to accumulate Zn, Ni and Cd in
excess of 1, 0.1, and 0.01% of dry weight, respectively are
considered as hyperaccumulators for these metals (Chaney
et al., 1997; Clemens, 2001). The metal hyperaccumulators
hold a high potential for being used in clean-up of toxic
metal-contaminated soils (phytoremediation). It appears
possible to engineer efficient plants for remediation purposes by combining the hyperaccumulation trait with the
high biomass production ability of crop species. A clearer
understanding of metal tolerance/hyperaccumulation mechanisms will greatly facilitate the realization of this goal.
Several transporters for micronutrients have been characterized which exhibit varying degrees of specificity, implying that non-essential metals share some of them for
entering the cell (Demidchik et al., 2002; Hall and
Williams, 2003). Furthermore, the vacuolar compartmentation of surplus metal concentrations seems to be a strong
component of the cellular metal detoxification strategy
(Dietz et al., 2001).
Upon exposure to metals, plants often synthesize a set of
diverse metabolites that accumulate to concentrations in the
millimolar range, particularly specific amino acids, such as
proline and histidine, peptides such as glutathione and
phytochelatins (PC), and the amines spermine, spermidine,
putrescine, nicotianamine, and mugineic acids. Thus,
nitrogen metabolism is central to the response of plants to
heavy metals. The scheme presented in Fig. 1 gives
examples addressed in the paper and the metabolic link.
Except for PC with metal-dependent activation of enzyme
activity, nicotianamine, and mugineic acid synthesis, the
responses may not or not in each case be the primary plant
reactions to heavy metals. However, from the data available, it has become clear that changes in the contents of
these metabolites bear functional significance in the context
of metal stress tolerance. Therefore, this review compiles
the information available on selected stress metabolite
accumulation under conditions of heavy metal exposure
and examines the functional significance of the response.
Emphasis has been given to proline that appears to possess
a unique function not only under drought but also in heavy
metal-stressed plants.
Proline
The proteinogenic amino acid proline functions as an
osmolyte, radical scavenger, electron sink, stabilizer of
macromolecules, and a cell wall component (Matysik
et al., 2002). Under salinity, proline accumulates to whole
tissue concentrations up to 1 mol lÿ1. Increased levels of
proline correlate with enhanced salinity tolerance (for a review see Munns, 2005), for example, in tobacco cell culture
expressing the regulatory Hal3 gene from Arabidopsis
thaliana (Yonamine et al., 2004).
Proline synthesis
Proline is predominantly synthesized from glutamate (Fig.
2). Three enzymatic activities, namely (i) the D1-c-glutamyl
kinase activity of D1-pyrroline-5-carboxylate synthetase
(At2g39800), (ii) the glutamic-c-semialdehyde dehydrogenase activity of D1-pyrroline-5-carboxylate synthetase
Amino acids and heavy metal stress
713
Fig. 1. Schematic depiction of synthetic pathways involved in the synthesis of amino acids and heavy metal-related N-metabolites in plants. The inner
area shows the central pathways of glycolysis and citric acid cycle and the linking metabolites to amino acid synthesis. Also shown is the branching to
glutathione (GSH), phytochelatin (PC), polyamine, nicotianamine, and mugineic acid (MA) synthesis. In the outer frame, examples are given showing
the involvement of N-metabolites in metal metabolism. HM: heavy metal.
(PCS), and (iii) two isogenes of D1-pyrroline-5-carboxylate
reductase (PCR) convert glutamate to proline in three
exergonic reactions consuming 1 ATP and 2 NADPH
per proline. The consumption of two moles of NADPH
implies that proline accumulation may serve as an electron sink mechanism. Alternatively, proline is generated
from ornithine by ornithine-d-aminotransferase, where D1pyrroline-5-carboxylate is produced (not shown). Transcript abundances in plant tissues of Arabidopsis thaliana
and A. halleri were derived from the NASC-arrays database
that contains microarray hybridization results from more
than 1000 experiments. Table 1 shows that strong transcript
regulation is reported for D1-pyrroline-5-carboxylate synthetase (P5CS1) in Arabidopsis thaliana roots upon treatment with lead, with induction factors (IF) of IF=6 at 25 lM
Pb and IF=31 at 50 lM Pb. Other transcripts involved in
proline synthesis only revealed weak up- and downregulation, for instance P5CS2 in leaves from Pb-treated
plants with factors of about 1.4. Transcripts of P5CS1 are
also up-regulated in response to Cs stress (Sahr et al.,
2005).
Accumulation
Proline is an extensively studied molecule in the context of
plant responses to abiotic stresses. Many plants accumulate
this compatible solute under water deficit (Aspinall and
Paleg, 1981), salinity (Ashraf and Harris, 2004), low
temperature (Naidu et al., 1991), high temperature, and
some other environmental stresses. Up-regulation of proline is often encountered in plants, ranging from algae to
angiosperms, under heavy metal stress too. Some reports
are listed in Table 2. Heavy metal-induced proline accumulation is generally comparable, in magnitude and other
characteristics, to that occurring under other abiotic
stresses. An increase of up to >20-fold in the free proline
content in the leaves of metal non-tolerant Silene vulgaris
was observed. Concentrations in the range of 10 to 50 mM
are commonly encountered. The proline-inducing ability
of different metals varied (Fig. 3). When compared at
equimolar concentrations in the growth medium, Cu was
most effective in inducing proline accumulation followed
by Cd and Zn, respectively. But, when compared at equal
toxic strength, proline accumulation decreased in the order
Cd > Zn > Cu (Schat et al., 1997). Cu and Cd proved to be
strong inducers of proline in other plant species as well
(Alia and Saradhi, 1991; Bassi and Sharma, 1993a, b). The
magnitude of the metal-induced increase in proline content
in other plant species was lower than that in Silene vulgaris,
although they were exposed to higher metal concentrations.
Often, the increase in shoot proline content is higher than
that in roots. Thus, the bulk of metal-induced proline in
Silene vulgaris was restricted to the leaves (Schat et al.,
1997). Nevertheless, the root proline content in Lactuca
sativa increased proportionally with an increase in Cd
concentration causing a 13-fold rise at 100 lM Cd (Costa
and Morel, 1994). Zn and Cu-induced proline rise in Lemna
minor was fairly rapid; it was measurable within 6 h of
metal treatment (Bassi and Sharma, 1993a). Similarly,
a Cu-dependent increase in the proline level of detached
rice leaves was detected within 4 h (Chen et al., 2001).
714 Sharma and Dietz
In Scenedesmus, Pro contents increased in response to Cu
and Zn (Tripathi and Gaur, 2004). In the case of Cu, Pro
contents peaked at 10 lM while it increased steadily with
Zn up to 100 lM.
High constitutive proline in metal-tolerant plants
The metal-tolerant populations of three different species
namely, Armeria maritima, Deschampsia cespitosa, and
Silene vulgaris have been reported to possess substantially
elevated constitutive proline levels in different plant parts
even in the absence of excess metal ions when compared
with their non-tolerant relatives. A highly Cu-tolerant
Armeria maritima population from a copper-containing
bog in Wales contained a high proline content in the roots.
The trait was heritable since plants derived from the seeds
collected from bog plants also exhibited similarly high
proline levels. By contrast, the plants from a non-copper
site did not show the elevated proline contents (Farago
and Mullen, 1979). Likewise, the Zn-tolerant clones of
Deschampsia cespitosa, grown on medium with nitrate as
the N source, contained about 4-fold increased levels of
proline in roots when compared with the non-tolerant
clones (Smirnoff and Stewart, 1987). Unlike Armeria
and Deschampsia, the elevated constitutive proline in
metal-tolerant Silene vulgaris, 5–6-fold over that in nontolerant plants, was predominantly restricted to the leaves
(Schat et al., 1997). Following the application of excess
metals, the responses of D. cespitosa and S. vulgaris
populations essentially resembled each other in that the
sensitive lines of both species accumulated high proline
contents but the tolerant ones did not (Smirnoff and
Stewart, 1987; Schat et al., 1997). The proline increase in
metal-tolerant S. vulgaris due to very high Cd concentrations was an exception. The decreased relative proline
accumulation in the tolerant ecotype could be due to
a slower development of water deficit under metal stress
(Schat et al., 1997).
Fig. 2. Synthesis of proline and participating enzymes.
Proline in metal tolerance
The occurrence of high constitutive proline contents in
metal-tolerant populations of three taxonomically unrelated
species is striking. It is tempting to assume a function for
proline in increased metal tolerance in these species. The
higher proline production has recently been demonstrated
to correlate with increased metal tolerance in a transgenic
alga (Siripornadulsil et al., 2002). In this study, a gene
encoding mothbean D1-pyrroline-5-carboxylate synthetase
(P5CS), responsible for catalysis of the first step of proline
synthesis, was introduced into the nuclear genome of green
microalga Chlamydomonas reinhardtii. The transgenic alga
produced 80% higher proline levels than the wild-type
cells, grew more rapidly in Cd concentrations as high as
100 lM, and bound 4-fold more Cd than the wild-type
cells.
Amino acids and heavy metal stress
715
Table 1. Transcript levels of enzymes involved in proline and polyamine synthesis
The data were extracted from the NASCArray database. The induction factor is given relative to the untreated control (in the absence of excess heavy
metal). The experimental conditions were: (i) Zn: Zn hyperaccumulating Arabidopsis halleri (A.h.) and non-tolerant Arabidopsis petraea (A.p.) grown
in full-strength medium supplemented with 25 or 50 lM Zn, (ii) Pb: Arabidopsis thaliana plants exposed to 25 or 50 ppm Pb(NO3)2, (iii) Cs: Arabidopsis
thaliana, 7 d at 2 mM Cs in 1/10 MS salt medium (Craigon et al., 2004).
Zn exposure
Description
A.h. roots, 25
A.h. leaves, 25
At5g14800
At5g46180
Pyrroline-5-carboxylate reductase
Ornithine aminotransferase,
putative/ornithine–oxo-acid
aminotransferase, putative
Spermidine synthase-related/putrescine
aminopropyltransferase-related
Ornithine aminotransferase,
putative/ornithine–oxo-acid
aminotransferase, putative
Spermine/spermidine synthase
family protein
Spermidine synthase 1
(SPDSYN1)/putrescine aminopropyltransferase 1
Spermidine synthase,
putative/putrescine aminopropyltransferase, putative
Spermidine synthase 2 (SPDSYN2)/putrescine
aminopropyltransferase 2
1.36
0.76
1.10
1.12
0.80
0.85
1.08
1.60
0.75
1.77
0.91
1.05
0.76
1.12
0.85
1.60
1.39
2.62
0.81
0.60
1.88
0.28
1.28
1.00
0.77
1.07
0.67
1.15
0.95
0.51
0.80
1.25
Pb exposure
Description
A.t. roots, 25
A.t. leaves, 25
A.t. roots, 50
A.t. leaves, 50
At3g55610
Delta 1-pyrroline-5-carboxylate
synthetase B/P5CS B (P5CS2)
Delta 1-pyrroline-5-carboxylate
synthetase A/P5CS A (P5CS1)
Spermine/spermidine synthase family protein
Spermidine synthase 1 (SPDSYN1)/putrescine
aminopropyltransferase 1
Spermidine synthase 2 (SPDSYN2)/putrescine
aminopropyltransferase 2
0.84
1.36
0.66
1.42
5.92
0.73
31.35
0.81
0.56
1.04
1.03
1.12
0.74
0.81
0.83
1.12
0.97
0.72
At5g04610
At5g46180
At5g19530
At1g23820
At5g53120
At1g70310
At2g39800
At5g19530
At1g23820
At1g70310
Cs exposure
Description
A.t. roots
A.t. shoot
At5g14800
At5g46180
Pyrroline-5-carboxylate reductase
Ornithine aminotransferase,
putative/ornithine-oxo-acid aminotransferase, putative
Acetylornithine aminotransferase, mitochondrial,
putative/acetylornithine transaminase,
putative/AOTA, putative
Spermidine synthase-related/putrescine
aminopropyltransferase-related
Spermine/spermidine synthase family protein
Spermidine synthase 1 (SPDSYN1)/putrescine
aminopropyltransferase 1
Spermidine synthase, putative/putrescine
aminopropyltransferase, putative
Spermidine synthase 2 (SPDSYN2)/putrescine
aminopropyltransferase 2
0.92
0.72
1.22
1.37
0.46
0.76
0.94
1.12
2.94
1.37
0.21
1.21
0.53
1.32
1.22
0.85
At1g80600
At5g04610
At5g19530
At1g23820
At5g53120
At1g70310
Mechanism of Proline accumulation
Proline accumulation may be realized by increased synthesis, release from macromolecules or decreased degradation. Costa and Morel (1994) suggested that inhibition
of proline oxidation is the reason for higher root proline
levels. Chen et al. (2001) found excess Cu-induced proline accumulation in detached rice leaves to be related to
proteolysis and increased activities of D1-pyrroline-5carboxylate reductase or ornithine-d-amino-transferase.
One of the consequences of exposure to heavy metals is
the deterioration of the plant–water balance (Rauser and
A.p. roots, 25
0.51
A.p. leaves, 25
0.76
Dumbroff, 1981; Poschenrieder et al., 1989; Barcelo and
Poschenrieder, 1990). This may trigger the accumulation of
proline, for example, in response to Cd in L. sativa (Costa
and Morel, 1994). Schat et al. (1997) established the
dependence of metal-imposed proline induction on the
development of water deficit. They found that suppression
of transpiration at raised relative humidity (98%), as
achieved by placing the plants under transparent polyethylene covers, inhibited proline accumulation almost
completely, even at metal accumulation rates in leaves
responsible for up to a 20-fold increase of leaf proline levels
716 Sharma and Dietz
Table 2. Selected reports describing heavy metal-induced
proline accumulation in plants
Plant species
Flowering plants
Cajanus cajan
Vigna mungo
Helianthus annuus
Lemna minor
Triticum aestivum
Lactuca sativa
Silene vulgaris
Oryza sativa
Algae
Anacystis nidulans
Chlorella sp.
Chlorella vulgaris
Scenedesmus sp.
Heavy metals
Reference
Cd, Co, Zn, Pb
Cd, Co, Zn, Pb
Pb, Cd, Cu, Zn
Zn, Cu
Zn, Cu
Cd, Co, Zn, Pb
Cd
Cd, Cu, Zn
Cu
Alia and Saradhi, 1991
Alia and Saradhi, 1991
Kastori et al., 1992
Bassi and Sharma, 1993a
Bassi and Sharma, 1993b
Alia and Saradhi, 1991
Costa and Morel, 1994
Schat et al., 1997
Chen et al., 2001
Cu
Cu
Cu
Cu/Zn
Wu et al., 1995
Wu et al., 1998
Mehta and Gaur, 1999
Tripathi and Gaur, 2004
Non-tolerant Silene vulgaris
Proline [µmol g-1 leaf dry weight]
20
Cu
Cd
Zn
10
0
Tolerant Silene vulgaris
20
Cd
10
Cu
0
0.1
Zn
1
10
100
1000
metal concentration [µM]
Fig. 3. Accumulation of proline in response to Cu-, Cd-, and Znexposure in Silene vulgaris. Modified from Schat et al. (1997) and is
reproduced by kind permission of Blackwell Publishing.
at 75% relative humidity in uncovered control plants. In
a converse manner, Kastori et al. (1992) concluded that
metal-induced proline accumulation in sunflower is a direct
consequence of metal uptake excluding a role of water
deficit. The conclusion was based on the observation that,
in their study, proline accumulation also occurred in isolated ‘fully turgid’ leaf discs floating on solution. However,
the metal concentrations used were sufficiently high to
damage the permeability functions of membranes causing
the loss of osmolytes and hence, turgor (De Vos et al.,
1991, 1993). Consequently, the role of water potential in
metal-induced proline accumulation was not ruled out.
The possibility of ABA mediating Cu-induced proline
accumulation in detached rice leaves has been suggested
(Chen et al., 2001). Light was identified as another factor
that promotes proline accumulation under environmental
stresses (Joyce et al., 1992) including Cd exposure (Arora
and Saradhi, 1995). The mechanism of light-dependent
stimulation is not understood.
Suggested functions of proline
Osmoregulation: Based on its known properties proline
may be involved in plant heavy metal stress by different
mechanisms, i.e. osmo- and redox-regulation, metal chelation, and scavenging of free radicals. The role of proline
in osmoregulation and, in turn, in conferring plants the
ability to withstand water deficit stress is well established
(Aspinall and Paleg, 1981; Kavikishor et al., 1995).
Transgenic tobacco containing elevated constitutive proline contents was less susceptible to the inhibitory effects
of severe osmotic stress (Kavikishor et al., 1995). In
addition to contributing to the osmotic adjustment at
relatively high concentrations, proline at low concentrations affected the expression of certain genes associated
with osmotolerance (Kiyosue et al., 1996). Thus, it is likely
that proline offsets the water deficit developed due to the
exposure to heavy metals. Proline has been reported to
bring about stomatal closure in several plant species (e.g.
Rajagopal, 1981). Such an effect would be expected to
restrict the metal uptake and translocation via suppression
of transpiration. The significance of a resultant reduction in
metal toxicity through this mechanism remains to be examined. Osmoregulation appears to be a common element
of plant reactions to various abiotic stresses. This implies
that the plant populations capable of efficient osmoregulation, such as the drought-tolerant ones, might better cope
with the component of metal toxicity that also includes
the development of water-deficit, for example, due to root
growth inhibition (Barcelo and Poschenrieder, 1990).
Proline-dependent metal chelation: The possibility of proline involvement in the chelation of metal ions is indicated.
Proline was demonstrated to protect glucose-6-phosphate
dehydrogenase and nitrate reductase in vitro against Znand Cd-induced inhibition. The measurements with a Cdspecific electrode revealed that the proline-dependent
enzyme protection was based on a reduction of free metal
ion activity in the assay buffer due to formation of a metal–
proline complex (Sharma et al., 1998). A role for proline–
enzyme or proline–water interactions such as the one
suggested in the case of proline-dependent protection of
enzyme activity against salinity, heat or dilution (Schobert
and Tschesche, 1978; Krall et al., 1989; Solomon et al.,
1994) was not evident in this experiment. The preliminary mass spectroscopic analyses made by the authors indicated the formation of proline–Cd complexes of variable
Amino acids and heavy metal stress
masses in an aqueous assay system (SS Sharma, M Georgi,
KJ Dietz, unpublished results). Regarding the significance
of the metal chelation function of proline in vivo, it is
interesting that Cu in the roots of Cu-tolerant A. maritima
was thought to exist as a Cu–proline complex (Farago and
Mullen, 1979). However, results contrary to this have also
been reported. The possibility that cytoplasmic free Pro
directly sequesters Cd was examined in the wild type and
a Pro-overproducing transgenic Chlamydomonas reinhardtii by determining the chemical identity of the atoms
binding Cd using extended X-ray absorption fine structure
(EXAFS) (Siripornadulsil et al., 2002). Data suggested that
Cd was not complexed by the same conjugates in the wild
type and transgenic algae. In P5CS-1-expressing transgenic
alga, the EXAFS spectrum was best fit by Cd co-ordination
to four S atoms suggesting Cd sequestration by phytochelatins and not Pro. By contrast, Cd was co-ordinated by
O and S atoms in the wild-type cells which was consistent
with the findings of Adhiya et al. (2002). For metals like
Cd, Pro chelation does not seem to be important as Cd
strongly induces phytochelatins (PCs) that are likely to
chelate most of the root Cd in short-term exposure
(DeKnecht et al., 1994). Wagner (1993) suggested citric
acid to be a major ligand for Cd at low cellular concentrations. Metal ligands may vary on an organ- and
compartment-specific basis even for the same metal. Thus,
Salt et al. (1995) found that Cd in Brassica juncea roots
was chelated by S-ligand (PCs). But in the xylem sap it was
involved with some, hitherto unknown, N- or O-containing
ligand. It will indeed be interesting to work out the relative
contribution of proline to metal chelation in a cellular
scenario, i.e. in the presence of co-occurring ligands.
Proline may be of particular importance in binding metal
ions that do not form complexes with PC. Currently,
emerging metabolomic approaches should be applied to
plants exposed to metal ions with diverse properties and
that might be rewarding in this context.
Proline as an antioxidant: Exposure of plants to both redox
active, for example, Cu and Hg, and other metals, for
example, Cd and Zn, induces the stimulated generation of
free radicals that leads to oxidative stress (De Vos et al.,
1991; Weckx and Clijsters, 1996, 1997; Dietz et al., 1999).
This represents one of the major causes of toxicity particularly due to redox metals. The cells are equipped with
an elaborate network of antioxidative enzymes and low
molecular weight metabolites which mitigate the oxidative
stress (Dietz et al., 1999). Proline has been reported to
scavenge different free radicals in certain in vitro generation
and detection systems. Smirnoff and Cumbes (1989)
demonstrated the hydroxyl radical (OH) scavenging property of proline. OH radicals were generated by ascorbate/
H2O2 or by xanthine oxidase/hypoxanthine/H2O2 and
detected by hydroxylation of salicylate or by denaturation
of malate dehydogenase. The reaction product formed
717
between proline and OH was not determined in this study.
Proline might react with OH under H+-abstraction by
forming a stable radical with spin on the C-5 atom (Rustgi
et al., 1977). A proline-nitroxyl radical R2N-O is also
known to be formed between Pro and OH (Floyd and
Nagi, 1984). Similarly, Alia et al. (2001) reported the singlet oxygen (1O2) quenching action of proline. They generated 1O2 photochemically by illumination of toluidine blue
and detected, through EPR spectroscopy, the 1O2-dependent
formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)
from 2,2,6,6-tetramethylpiperidine (TEMP). The 1O2
quenching of proline seems to be based on its capability
to form a charge-transfer complex due to a low ionization
potential. Further evidence for in vitro proline-dependent
free radical scavenging has been obtained from graft copolymerization reactions. The latter are performed in
polymer chemistry and involve grafting of some monomer
such as methyl acrylate onto a polymeric backbone such
as cellulose. The grafting is essentially mediated by free
radicals generated via a chemical or a physical initiator and
could be taken as a quantitative measure of free radical
generation. Inclusion of proline in the reaction system
substantially inhibited the extent of grafting initiated either
by Ce4+ ions or c-radiation (S Kaul, IJK Mehta, SS
Sharma, unpublished results). Proline did not interact
with superoxide radicals (Smirnoff and Cumbes, 1989).
The mechanisms of ROS quenching by proline have been
summarized recently by Matysik et al. (2002).
Proline possibly detoxifies the ROS under stress in vivo
too (Smirnoff, 1993). The pretreatment of Chlorella
vulgaris with exogenous proline was found to counteract
lipid peroxidation as well as K+ efflux observed after
exposure to the heavy metals Cu, Cr, Ni, and Zn. Proline
accumulation in C. vulgaris due to metal treatment was also
evident (Mehta and Gaur, 1999). Similarly, proline, added
to the nutrient medium, lowered the inhibitory influence of
Cu on the growth of cyanobacterium Anacystis nidulans;
the effect being more pronounced when proline was added
prior to Cu treatment (Wu et al., 1995). The effect was
explained in terms of proline-dependent protection to the
membranes against Cu-induced K+ leakage. Later, Wu
et al. (1998) reported reduced Cu uptake by Chlorella cells
containing high proline concentrations. Free proline content
correlated positively with Cu tolerance of the lichen
photobiont Trebouxia erici (Backor et al., 2004). The toxic
metal effects were also alleviated by proline in Scenedesmus armatus (El-Enany and Issa, 2001). Recently, free
proline levels have been found to be correlated with the
GSH redox state and MDA content in Cd-treated low (wild
type) and high (transgenic) proline strains of C. reinhardtii
(Siripornadulsil et al., 2002). Thus, the wild-type cells
exhibited a 4-fold more oxidized glutathione pool than
transgenic P5CS-1 expressing cells when grown in the
presence of Cd suggesting a less oxidizing cytoplasmic
state in transgenic than in the wild-type cells under Cd
718 Sharma and Dietz
stress. Also, the transgenic cells produced much less MDA
than the wild-type cells upon exposure to Cd. Based on
these findings, free Pro was suggested to act as an
antioxidant in Cd-stressed cells; consequently, increased
GSH levels favour enhanced phytochelatin synthesis and
sequestration of Cd.
In Scenedesmus, Pro pretreatment decreased lipid peroxidation induced by 5 lM Cu by more than 75% similar
to methylviologen- and UV-B-induced MDA generation,
while Zn-dependent lipid peroxidation was suppressed by
30% only (Tripathi and Gaur, 2004). These data concur
with a preferential antioxidant function of Pro by detoxifying hydroxyl radicals making the metal binding unlikely.
Recently, metal-dependent activation of mitogen-activated
kinase pathways has been demonstrated in plants following Cd and Cu treatment (Jonak et al., 2004). The data
indicate the differential and metal-specific activation of
signalling pathways and the significance of ROS in mediating the stress responses. In this context it will also be important to dissect the diverse possible functions of proline in
metal-stressed plants.
Proline as regulator: Specific Pro functions in plant morphogenesis were revealed in antisense transgenic Arabidopsis
with decreased levels of pyrroline-5-carboxylate synthetase. In parallel with decreased levels of Pro, the plants
revealed morphological alterations in leaves and a defect in
inflorescence elongation. Structural cell wall proteins were
specifically affected in the antisence transgenics (Nanjo
et al., 1999). Based on a parallelism between Cd-induced
growth inhibition and a rise in proline, Chen and Kao
(1995) suggested proline to mediate the toxic effect of Cd
on rice seedlings. The exogenous proline mimicked the Cd
effect. In fact, there are a few recent reports indicating the
toxicity of exogenous Pro in plants (Hellmann et al., 2000;
Deuschle et al., 2001). Nanjo et al. (2003) evaluated the
Pro toxicity in Arabidopsis T-DNA tagged mutant pdh that
was defective in Pro dehydrogenase (AtProDH), responsible for catalysing the first step of Pro catabolism. The
pdh mutant was hypersensitive to exogenous L-Pro at
concentrations <10 mM while wild-type plants grew
normally at such concentrations. In a recent study by
Yamada et al. (2005) exogenous Pro-dependent growth
inhibition and Pro accumulation in Arabidopsis thaliana
were lower than that in petunia indicating the speciesspecificity of the phenomenon. Further research towards
integration of the growth inhibiting and protecting properties of Pro is needed.
production (Krämer et al., 1996). Whereas the exposure
to Ni resulted in a large and proportionate increase in histidine concentration in xylem sap in Ni-hyperaccumulating
A. lesbiacum, such a response was lacking in the nonaccumulator A. montanum. The significant co-ordination
of Ni with histidine was revealed in the extended X-ray
absorption fine structure analysis of the xylem sap in A.
lesbiacum. Thus, Ni-hyperaccumulation relies on histidinedependent root-to-shoot translocation of Ni. Furthermore,
A. montanum, when supplied with equimolar concentrations of exogenous histidine, transported more Ni to the
shoot as a result of enhanced Ni flux into the xylem and
showed tolerance to applied Ni concentrations. Similar
results have been obtained with Ni non-accumulator
Brassica juncea (Kerkeb and Krämer, 2003). The constitutive His contents of A. lesbiacum roots were 4.4-fold
higher than that in B. juncea. Kerkeb and Krämer (2003)
examined the role of free His in xylem loading in A.
lesbiacum and B. juncea. The data are consistent with
a model where Ni uptake is independent of His uptake and
the enhanced release of Ni into the xylem is associated with
a concurrent release of His from an increased root free His
pool. Another Ni-hyperaccumulator Thlaspi goesingense
exhibited increased root His concentrations when compared
with T. arvense. However, none of the three genes of
histidine biosynthesis studied was regulated by Ni (Persans
et al., 1999). Besides Ni, Salt et al. (1999) identified Zn–
His complexes in the roots of Zn-hyperaccumulator Thlaspi
caerulescens. Histidine levels have also been demonstrated
to correlate with tolerance to Ni and other metals of the
yeast Saccharomyces cerevisiae (Pearce and Sherman,
1999). Recently, a cell surface-engineered yeast displaying
a histidine oligopeptide (hexa-His) has been shown to
adsorb 3–8 times more copper ions than the parent strain
and to be more resistant to Cu than the parent (Kuroda
et al., 2001). The beneficial role of high histidine levels has
been shown in transgenic Arabidopsis thaliana expressing
a Salmonella typhimurium ATP phosphoribosyl transferase
enzyme (StHisG). The bacterial enzyme is insensitive to
feedback inhibition by histidine. The transgenic plants
accumulated about 2-fold higher histidine levels than wildtype plants and showed more than 10-fold increased
biomass production in the presence of toxic Ni in the
growth medium (Wycisk et al., 2004) convincingly demonstrating a mechanistic link between His and moderate Ni
tolerance.
Other amino acids
Histidine
Nearly three-quarters of all known metal hyperaccumulator
plants are Ni accumulators. The Ni-hyperaccumulation trait
in Alyssum species (Brassicaceae) has been demonstrated
to be specifically linked to the ability for free histidine
Zinc toxicity, in terms of root growth inhibition, in
Deschampsia cespitosa (metal tolerant and non-tolerant
clones) varied with the source of N in the growth medium.
Zn was invariably less toxic when N was supplied as
ammonium rather than nitrate. The ammonium-grown
Amino acids and heavy metal stress
non-tolerant clone was found to accumulate asparagine in
the roots; subsequently formed Zn-asparagine complex
may reduce Zn toxicity (Smirnoff and Stewart, 1987).
Properties of asparagine as ligand towards Cd, Pb, and Zn
have been reported from in vitro studies (Bottari and Festa,
1996). The accumulation and employment of specific amino
acids in metal tolerance and hyperaccumulation is a speciesspecific trait. Homer et al. (1997) determined the amino
acids in three species of Philippine Ni-hyperaccumulators
namely, Walsura monophylla, Phyllanthus palwanensis,
and Dechampetalum geloniodes. The Ni content in the
leaf extracts of these plants was 23.7, 34.3, and 134 lmol
gÿ1, respectively. Among all amino acids Pro was highly
abundant in all cases. Thus, the above species contained
29, 14.3, and 3 lmol gÿ1 proline, respectively. As an exception, glutamine was also present in high concentration
(29.8 lmol gÿ1) in W. monophylla. These findings indicate
that association between amino acids and metals could be
important particularly because Ni complexes with amino
acids are considerably more stable than those with carboxylic acids (Homer et al., 1997). The Ni-hyperaccumulator
Stackhousia tryonii was analysed for changes in amino
acid contents of xylem sap when grown at low or high Ni
(Bhatia et al., 2005). Total amino acid concentrations
slightly decreased from 22 to 18 mM and the proportion
of Gln declined from 48% to 22 mol% while Ala, Asp, and
Glu increased. Asp and Ala may contribute to complexation of Ni in xylem (Bhatia et al., 2005).
Zn administration to tomato and soybean strongly
affected amino acid concentrations (White et al., 1981a).
A study of metal complex equilibria in xylem fluids
suggests that the major portion of Cu and Ni was bound
to asparagine and histidine (White et al., 1981b). Also
present in exudates from high Zn-treated tomato were Cu–
aspartate and Cu–threonine complexes. Amino acids do not
bind Zn efficiently at acidic pH. Thus, organic acids are the
predominant ligands at low pH, but amino acid–Zn complexes are increasingly formed as the pH increases (White
et al., 1981b). Cysteine contents increased by factors of
4.5 and 3.8 in tolerant and sensitive ecotypes of Silene
vulgaris, respectively, in response to metal stress that
caused similar inhibition of root growth (Harmens et al.,
1993). Cysteine is required for methionine and glutathione/
phytochelatin synthesis, and, therefore, is a central metabolite in antioxidant defence and metal sequestration.
Genetically increased capacity for metal-induced Cys
synthesis was shown to support survival of Arabidopsis
under acute Cd stress (Dominguez-Solis et al., 2004). The
significance of these alterations in amino acid composition
for metal binding in vivo is difficult to assess. However, it
should be kept in mind that amino acids are asymmetrically
distributed between plasmatic and secretory compartments.
In barley mesophyll cells, the concentrations of total amino
acids in the extravacuolar compartment were about twice
that of the vacuole and may be in the range of 100 mmol
719
ÿ1
l ; similar values were likewise observed in chloroplasts
(Krause et al., 1984; Dietz et al., 1990). Such high concentrations further increase under stress and are likely to
contribute to heavy metal binding.
Polyamines
Polyamines are ubiquitous in all organisms. They influence
a variety of growth and development processes in plants
and have been suggested to be a class of plant growth
regulators and to act as second messengers (Evans and
Malmberg, 1989; Slocum and Flores, 1991; Kakkar and
Sawhney, 2002). The polyamines are cations due to protonation at cytoplasmic pH, i.e. putrescine2+, spermidine3+,
and spermine4+. This accounts for their binding ability to
nucleic acids (Flink and Pettijohn, 1975) and affinities for
phospholipids in plasmamembranes (Grimes et al., 1986).
The levels of polyamines and the activities of their biosynthetic enzymes in plants increase under environmental
stresses (Evans and Malmberg, 1989). Polyamines are synthesized from ornithine, citrulline or arginine following
decarboxylation to yield putrescine and the addition of one
or two aminopropyl groups from S-adenosyl methionine to
generate spermine and spermidine (Kakkar and Sawhney,
2002). Polyamine functions in plants are only slowly
unravelled. Transgenic and mutant plants with altered
polyamine levels reveal strong developmental aberrations
(reviewed in Kakkar and Sawhney, 2002). In addition,
polyamines have an antioxidative property by quenching
the accumulation of Oÿ
2 probably through inhibition of
NADPH oxidase (Papadakis and Roubelakis-Angelakis,
2005).
Polyamine contents are altered in response to the
exposure to heavy metals. Weinstein et al. (1986) showed
an up to 10-fold increment in putrescine content in Cdtreated oat seedlings and detached oat leaves with a
marginal rise in spermidine and spermine content. The
Cd-dependent increase in putrescine was blocked by difluoromethylarginine (DFMA), an inhibitor of arginine
decarboxylase (ARGdc), linking this enzyme with Cdinduced putrescine biosynthesis. The response of different
polyamines to Cd treatment strongly varied in Phaseolus
vulgaris in an organ-specific manner. Putrescine increased
in root, hypocotyl, and epicotyl whereas spermidine increased in hypocotyl, decreased in leaves, and did not
change in roots. By contrast, spermine level decreased in
all seedling parts. Al-induced changes in putrescine of
Catharanthus roseus were influenced by the cell age. Al
addition to the suspension cultures induced an increase in
putrescine level within 24 h in freshly transferred cells
but reduced the same at later stages (2–7 d) (Zhou et al.,
1995). Elevated putrescine and 1,3 diaminopropane (DAP)
due to Hg treatment were also observed in the green alga
Chlorogonium elongatum (Agrawal et al., 1992).
720 Sharma and Dietz
Transcripts encoding polyamine synthetic genes were
non-responsive or slightly up- and down-regulated in
response to Zn-, Pb-, and Cs-exposure (Table 1). For
example, spermine/spermidine synthase mRNA was increased with IF=2.6 in leaves of Arabidopsis halleri
exposed to 25 lM Zn, and with IF=2.9 in roots of Csstressed Arabidopsis thaliana. However, there was no
uniform response of the tissues to metal stressors.
A specific role of polyamines in plants under metal stress
is not yet known. However, there is a strong possibility that
they can effectively stabilize and protect the membrane
systems against the toxic effects of metal ions particularly
the redox active metals. Data supporting this are available
both from in vivo and in vitro studies. Thus, NADH- and
ascorbic acid-dependent lipid peroxidation in rat liver
microsomes was inhibited by polyamines; spermine was
most effective. Spermine binding to membrane phospholipids explained the observed inhibition (Kitada et al., 1979).
Using membrane vesicles prepared from mixed soybean
phospholipids, Tadolini et al. (1984) showed that polyamines inhibit lipid peroxidation when bound to the
negative charges on the vesicle surface. A pretreatment of
sunflower leaf discs with exogenous spermine was recently
found to reverse almost completely the Cd- or Cu-induced
lipid peroxidation (thiobarbituric acid reactive material)
(Groppa et al., 2001). It is important here that the isolated
plasmamembranes from root and hypocotyl of Cd-treated
mungbean seedlings contained greater polyamine amounts
than those from controls (Geuns et al., 1992). In addition,
polyamines namely, spermine, spermidine, putrescine, and
cadaverine have been demonstrated to scavenge free
radicals in vitro (Drolet et al., 1986). Furthermore, polyamines block one of the major vacuolar channels, the fast
vacuolar cation channel, and their accumulation could
decrease ion conductance at the vacuolar membrane to
facilitate metal ion compartmentation (Brüggemann et al.,
1998).
Glutathione and phytochelatins
The significance of glutathione and the metal-induced
phytochelatins (PCs) in heavy metal tolerance has been
summarized intensely in excellent reviews (Rauser, 1995,
1999; Hall, 2002). Depletion of glutathione appears to be
a major mechanism in short-term heavy metal toxicity
(Schützendübel and Polle, 2002). In accordance with this
hypothesis, a good correlation between glutathione contents and tolerance index was observed with 10 pea genotypes differing in Cd sensitivity (Metwally et al., 2005).
Freeman et al. (2004) reported the concentrations of glutathione and also Cys and O-acetyl-L-serine (OAS) in shoot
tissue to correlate strongly with the Ni-hyperaccumulation
ability of various Thlaspi hyperaccumulator species. High
GSH concentrations in hyperaccumulator T. goesingense
coincided with high constitutive activity of serine acetyl
transferase (SAT); SAT catalyses the acetylation of L-Ser
to OAS which in turn provides the carbon skeleton for
Cys biosynthesis. Elevated GSH levels in T. goesingense
also coincided with the ability both to hyperaccumulate Ni
and to resist its damaging oxidation effects. Further, the
arsenate hypertolerance in Aspergillus sp. P37 has recently
been linked to an enhanced capacity to maintain a large
intracellular glutathione pool under conditions of As
exposure and to sequester As(GS)3 in vacuoles. As-induced
formation of vacuoles with accumulated thiol species was
observed (Canovas et al., 2004). In addition to its function as an antioxidant, metal-dependent activation of PC
synthase puts strain on the glutathione pool. PCs are the
best known example of metal-induced metabolite accumulation and are implicated in cellular metal detoxification
(for review see Steffens, 1990; Rauser, 1995). They are
thiol-rich low molecular weight peptides synthesized by
plants in response to exposure to heavy metals; synthesis
from GSH is catalysed by phytochelatin synthase. The PC–
metal complex is often sequestered in the vacuoles. From
a variety of experimental approaches the involvement of
PCs in the detoxification of Cd and As (Cobbett, 2000,;
Schmöger et al., 2000) appears definite as they could also
be linked to the differential heritable tolerance to Cd in
Arabidopsis thaliana (Howden et al., 1995) and As in
Holcus lanatus (Hartley-Whitaker et al., 2001). However,
PCs are important for detoxification of only a limited set of
2+
2+
metals such as Cd2+, Cu2+ and AsO2ÿ
2 while Zn and Ni
are poor inducers of PCs and exhibit low binding affinity.
Most other metals lack significant binding at all. Although
overexpression of PC synthase (AtPCS1) in S. cerevisiae
did not alter the sensitivity to Zn and Ni, it induced an
enhanced tolerance to Cd, As, and Hg (Vatamaniuk et al.,
1999). There seems to be no kingdom barrier as to the role
of PCs in metal detoxification. Thus, Caenorhabditis
elegans was recently demonstrated to express a gene coding
for a functional phytochelatin synthase (PCS). Further
CePCS complemented the Cd sensitivity of a S. pombe
PCS knock-out strain by conferring the PCS activity
(Clemens et al., 2001; Vatamanuik et al., 2001). PCS is
post-translationally activated by metal binding to specific
activating sites (Maier et al., 2003). In addition to this
mechanism, recent work has shown that transcriptional
activation of PCS is another level of regulation that is
dependent on isoform, developmental stage, and growth
condition (Lee and Korban, 2002; Finkemeier et al., 2003).
In barley, mRNA levels of PCS were stimulated in response to Cd when the plants were low in nitrogen, pointing to an N-saving mechanism at low N (Finkemeier
et al., 2003). PCS protein accumulation in leaves was
also seen in Brassica juncea treated for 6 d with 25 lM
Cd, but the response was absent from roots (Heiss et al.,
2003). On the other hand, not all plants respond to Cd
with PC synthesis. In Salix treated with Cd, phytochelatins
Amino acids and heavy metal stress
(c-(Glu-Cys)nGly; n=2–3) were not detectable, suggesting
that heavy metal stress defence in Salix is independent of
the phytochelatins PC2 and PC3 (Landberg and Greger,
2004). Despite this surprising result, a central but not
exclusive role of phytochelatins in specific metal tolerance
is apparent.
Other N-containing metabolites in relation to
metal stress
Higher plants also synthesize other N-containing amino
acid-derived metabolites upon metal stress such as betaines,
mugineic acid, and nicotianamine. While betaines represent
general stress metabolites, the other two have a specific
function in metal homeostasis and metal stress defence.
Glycinebetaine, the trimethyl glycine, is a commonly
encountered osmolyte that accumulates in plants under
salinity and drought stress. Its synthesis predominantly
depends on decarboxylation of serine to ethanolamine that
is subsequently methylated with S-adenosylmethionine as
the methyl donor. Armeria maritima ecotypes were collected from sandy grassland, Zn-contaminated sites or salt
marshes (Köhl, 1996). Upon exposure to drought, the
plants from the Zn-mines accumulated more proline, but
also more betaine, than plants from sites without metal
contamination.
Nicotianamine (NA) is ubiquitously present in the plants
and is synthesized from three molecules of methionine by
nicotianamine synthase (NAS) (Fig. 4). The Arabidopsis
thaliana genome contains four nas genes. Initially, nicotianamine was identified in the context of Fe nutrition.
Three carboxylic acid groups within each molecule enable
high efficiency binding of Fe and other transition metals.
NA is a precursor for secreted mugineic acids, chelates Fe,
and participates in Fe transport (Schmidt, 2003). Ectopic
overexpression of nicotianamine aminotransferase (NAAT)
in tobacco, which synthesizes NA but not phytosiderophores, consumed endogenous NA and caused a severe
decrease in Cu>Fe>Zn>Mn contents in leaves to levels of
element deficiency and reproductive sterility (Takahashi
et al., 2003). Grafting of naat-tobacco shoots on wild-type
roots substantially restored Fe, Cu, and Zn concentrations
in naat-tobacco and sterility was overcome. From the data
the authors proposed multiple functions of nicotianamine in
the delivery of metals, particularly at the reproductive stage
(Takahashi et al., 2003). Recent work indicates that NA is
also important in heavy metal detoxification. The Znhyperaccumulator plant Arabidopsis halleri expresses the
nas-2 and -3 genes at a very high constitutive level, for
example, nas-2 with a 73-fold higher transcript level
compared with A. thaliana (Weber et al., 2004). The
response of nas 1–4 transcripts to elevated Zn supply was
not uniform, neither in the sensitive A. thaliana and
A. halleri, nor between roots and leaves (Becher et al.,
2004). Heterologous expression of Atnas2 in the yeast
721
Schizosaccharomyces pombe conferred increased Zn tolerance with a shift of IC50 from 50 lM in the wild type to
300 lM in the mutant yeast strain (Weber et al., 2004).
The data suggest an important role of NA in metal
homeostasis especially of hyperaccumulating species and
possibly in hyperaccumulation. NA could be involved in
the transfer of excess metals from the roots to the shoots.
Plants with decreased and increased NA contents should
be tested for altered metal tolerance.
Mugineic acids are synthesized in graminaceous species
from NA by transamination, oxidation, and hydroxylation
reactions (Kobayashi et al., 2005) and, thus, are based on
methionine building blocks as well (Fig. 4). They belong to
the group of phytosiderophores that are secreted under iron
deficiency to promote iron acquisition from the rhizosphere. Nicotianamine aminotransferase (NAAT) catalyses
the transfer of amino group from NA for the biosynthesis of
phytosiderophores. In addition to mobilizing Fe, phytosiderophores appear to affect heavy metal solubility, leading
to an increased bioavailability, for instance, of Cd (Collins
et al., 2003). Recently, the H+-symporter ZmYS1 has been
identified in maize that transports metal complexes of NA
and phytosiderophores. The KM-values were 19 lM for
deoxymugineic acid–Ni-complex and 165 lM for NA–Nicomplex (Schaaf et al., 2004). The transporter also carried
Cu-, Fe-, and Zn-complexes following its expression in
yeast. Since eight homologues of ZmYS1 have been
identified based on sequence similarity in the genome of
Arabidopsis thaliana, a plant that lacks phytosiderophore
production, it is hypothesized that these transporters may
be involved in metal ion distribution in general (Curie
et al., 2001; Schaaf et al., 2004). Mugineic acid certainly
needs more attention towards its role in metal delivery and
distribution under conditions of excess metals particularly
on Fe-limited soils.
Summary and outlook
The compiled data demonstrate the significance of nitrogencontaining metabolites beyond phytochelatins and glutathione in plant response and acclimation to heavy metals.
Since the various metal ions have specific chemical properties and induce distinct responses of adaptation and
damage development, it is not surprising that accumulating
N-metabolites display a variety of functions, i.e. metal ion
chelation, antioxidant defence, protection of macromolecules, and possibly signalling. The case of proline exemplifies the functional diversification of a compound initially
addressed as a compatible solute in the context of osmotic
and salinity stress: Proline is suggested to quench ROS
and reactive nitrogen species (RNS) and to relieve the oxidative burden from the glutathione system. This may facilitate phytochelatin synthesis and enhance metal tolerance
(Siripornadulsil et al., 2002). Direct metal binding by
Pro is suggested although a thorough evaluation of this
722 Sharma and Dietz
Fig. 4. Synthesis of nicotianamine and mugineic acid from methionine. Modified from Kobayashi et al., 2005 and reproduced by kind permission of
Oxford University Press.
function in vivo is required. It is difficult to dissect the
distinct contributions of individual functions of a compound
from the overall tolerance, particularly if the compound is
essential for basic metabolism. In a more straightforward
manner, current research may focus on the identification of
novel players in metal tolerance by employing postgenomic approaches such as metabolite profiling and
cDNA array analyses (Becher et al., 2004; Weber et al.,
2004; Urbanczyk-Wochniak and Fernie, 2005). The Fe
deficiency-induced up-regulation of all genes of the methionine cycle and nicotianamine synthesis represents an
intriguing and convincing example of the power of this
kind of approach (Kobayashi et al., 2005). This Fe-related
metabolism is directly linked to Zn stress and tolerance
as judged by the up-regulation of nicotianamine synthase
transcripts in cDNA array analyses with Arabidopsis
thaliana and Arabidopsis halleri (Becher et al., 2004;
Weber et al., 2004). On the other hand, mutants need to be
checked routinely for altered metal sensitivity. Such reverse
genetic approaches will certainly elucidate novel regulatory
and metabolic relationships, such as, for example, the role
of a mitochondrial Prx IIF (Finkemeier et al., 2005).
Insertional inactivation of Prx IIF gene had no effect on
Arabidopsis under optimum conditions, but growth was
severely impaired under stress such as Cd. It would not
be surprising if the list of N-metabolites involved in
metal homeostasis and heavy metal defence expands in
the near future.
Acknowledgements
Expert help from Miriam Hanitzsch in the NASC array analysis is
gratefully acknowledged. This review and parts of the own cited
work originate from a collaboration funded by the Indian National
Science Academy (INSA, India) and the DFG as well as DAAD
(Germany).
Amino acids and heavy metal stress
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