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Chapter 7 Nitrogen and Sulfur Metabolism in C4 Plants

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 Chapter 7
Nitrogen and Sulfur Metabolism in C4 Plants

Stanislav Kopriva*
John Innes Centre, Norwich NR4 7UH, UK

Summary ............................................................................................................................................................... 109

I. Introduction..................................................................................................................................................... 110
II. Nitrogen Assimilation...................................................................................................................................... 110
A. Plant Nitrate Assimilation ......................................................................................................................... 110
B. Regulation of Nitrate Assimilation ............................................................................................................ 111
C. Nitrate Assimilation in C4 Plants ............................................................................................................... 113
III. Sulfate Assimilation ........................................................................................................................................ 114
A. Plant Sulfate Assimilation ......................................................................................................................... 114
B. Regulation of Sulfate Assimilation ............................................................................................................ 115
C. Sulfate Assimilation in C4 Plants .............................................................................................................. 116
IV. Glutathione Synthesis and Reduction ............................................................................................................ 117
A. Regulation of GSH Synthesis ................................................................................................................... 117
B. Localization of GSH and GSH Synthesis ................................................................................................. 118
C. GSH Synthesis in C4 Plants ..................................................................................................................... 119
V. Physiological Significance of the Distribution of Nitrate and Sulfate Assimilation .......................................... 120
A. Open Questions on Nitrate Assimilation in C4 Plants ............................................................................... 120
B. Significance of BSC Localization of Sulfate Assimilation ......................................................................... 121
C. Consequences of BSC Localization of Sulfate Assimilation..................................................................... 122
VI. Conclusions .................................................................................................................................................... 122
Acknowledgments ................................................................................................................................................. 122
References ............................................................................................................................................................ 123

Summary

C4 photosynthetic mechanism is based on a spatial separation of CO2 assimilating enzymes. The assimila-
tion of two mineral nutrients, nitrogen and sulfur, is also localized in a cell-specific manner in most C4
species. N assimilation seems to be confined to mesophyll whereas sulfate reduction has been previously
reported to be bundle sheath specific. The latter view has been challenged by finding an ubiquitous pres-
ence of enzymes of sulfate assimilation in the dicot C4 species of Flaveria. Although inter- and intracellular
distribution of enzymes of N assimilation in C4 plants differ from C3 plants and C4 plants have a better N
use efficiency, very little is known about the physiological consequences of this distribution. Analogically,
no evolutionary advantage for the BSC localization of sulfate assimilation has been identified. On the other
hand, the organization and general regulation of the pathways is the same in C3 and C4 plants. In this chapter
the two essential pathways of plant primary metabolism, nitrate and sulfate assimilation, as well as the syn-
thesis of glutathione, the major sulfur containing metabolite involved in stress defense, will be described.
The general regulation of the pathways as well as specific features connected with C4 photosynthesis
will be discussed. The major open questions of N and S metabolism in C4 plants will be addressed.

*Author for Correspondence, e-mail: stanislav.kopriva@bbsrc.ac.uk

Agepati S. Raghavendra and Rowan F. Sage (eds.), C4 Photosynthesis and Related CO2 Concentrating Mechanisms, pp. 109–128. 109
© Springer Science+Business Media B.V. 2011
110
I. Introduction
A characteristic feature of C4 plants is a cell-
specific localization of many enzymes of pri-
mary metabolism in bundle sheath cells (BSC) or
mesophyll cells (MC) (for details see Chapter 12,
this volume). Clearly, the enzymes involved in the
primary CO2 fixation and malate and/or aspartate
synthesis, such as cytosolic carbonic anhydrase,
phosphoenolpyruvate carboxylase, pyruvate phos-
phate dikinase, and NADP-malate dehydrogenase,
are localized predominantly in the MC, whereas
NAD(P)-malic enzyme, Rubisco, Rubisco acti-
vase, and some enzymes of the Calvin cycle are
found exclusively in BSC (reviewed in Sheen,
1999; Edwards et al., 2001). In most C4 species
analyzed, including maize and Sorghum, BSC
chloroplasts lack photosystem II and therefore
exhibit very little oxygen evolution (Hatch and
Osmond, 1976). Consequently, noncyclic electron
flow and the capacity for NADPH formation are
restricted in BSC chloroplasts. In addition, glycine
decarboxylase, a key enzyme of photorespiration,
is localized exclusively in BSC of C4 and C3-C4
intermediate plants (Hylton et al., 1988; see Chap-
ter 6, this volume). The C3–C4 intermediate plants
were originally identified by having a CO2 com-
pensation point intermediate between C3 and C4
species. They can be considered as evolutionary
intermediates in the path from C3 to C4 photosyn-
thesis (Monson and Moore, 1989; Kopriva et al.,
1996). The intermediate species possess a Kranz-
like anatomy and their apparent photorespiration
rate is reduced, due to the confinement of glycine
decarboxylase to the BSC and efficient refixation
of photorespired CO2 in these cells (Hylton et al.,
1988; Rawsthorne, 1992; cf. Chapter 6, this vol-
ume). Some C3–C4 plants are to some extent able
to fix CO2 into malate and aspartate as C4 species
(Bassüner et al., 1984; Monson et al., 1986), but
Abbreviations: APR – Adenosine 5¢-phosphosulfate reductase;
APS – Adenosine 5¢-phosphosulfate; ATPS – ATP sulfury-
lase; BSC – Bundle sheath cells; GECg-Glutamylcysteine;
GECSg-Glutamylcysteine synthetase; GOGAT – Glutamate
synthase; GR – Glutathione reductase; GS – Glutamine
synthetase; GSH – Glutathione; GSHS – Glutathione syn-
thetase; GSSG – Oxidized glutathione, glutathione disulfide;
MC – Mesophyll cells; NR – Nitrate reductase; OAS – O-
Acetylserine; ROS – Reactive oxygen species;
Stanislav Kopriva
the compartmentalization of the photosynthetic
enzymes is not complete (Bauwe, 1984).
Interestingly, apart from enzymes involved in
the C4 carbon cycle enzymes participating in the
assimilation of nitrogen and sulfur are also local-
ized in cell-specific manner in C4 plants. In this
chapter, the two pathways will be described in
detail with the focus on their subcellular distribu-
tion and the consequence of such distribution for
the regulation of the pathways and for the per-
formance of C4 plants.
II. Nitrogen Assimilation
Nitrogen (N) is the most abundant mineral nutri-
ent in plant tissues but also the element most
frequently limiting plants growth (Vance, 2001).
The major N sources are inorganic nitrate and
ammonium, however, plants developed several
other strategies to meet their demand for N. The
best known alternative source of N for plant
nutrition are the symbiotic N2 fixing root nodules
(Day et al., 2001) Also mycorrhizal fungi have
been shown to contribute to plant N acquisition
(Chalot and Brun, 1998), as well as N2 fixing bac-
teria in the phyllosphere (Papen et al., 2002). In
addition, uptake of organic N compounds, such
as amino acids, may cover substantial part of
N acquisition in certain habitats (Persson et al.,
2003). C4 plants are capable of mycorrhiza sym-
biosis; however, no C4 species form nodules with
symbiotic bacteria.
A. Plant Nitrate Assimilation
The most common form of N plants acquire from
the soil is nitrate. Nitrate is transported into plant
cells by nitrate transporters (Fig. 1). Three uptake
systems are responsible for uptake of nitrate into
the roots: a constitutive high affinity uptake sys-
tem, an inducible high affinity system an a low
affinity system (Miller et al., 2007). However,
further transporters are necessary to facilitate
xylem loading and distribution of nitrate through-
out the plant as well as its storage in the vacuoles.
Two major classes of nitrate transporters exist in
plants, the NRT1/PRT family responsible for low
affinity nitrate uptake as well as amino acid and
peptide transport, which contains 53 genes in
Arabidopsis, and NRT2 family of high affinity
7 Nitrogen and Sulfur in C4 Plants
Fig. 1. Schematic representation of plant nitrate assimilation.
Dark shaded rectangle represents mitochondria, light shaded
one denotes plastid. Enzymes are symbolized by numbers: 1 –
nitrate transporter, 2 – ammonium transporter, 3 – nitrate
reductase, 4 – nitrite transporter, 5 – nitrite reductase, 6 –
glutamine synthetase, 7 – glutamate synthase, 8 – plastidic
glutamate–malate translocator, 9 – plastidic 2-oxoglutarate–
malate translocator, 10 – mitochondrial glutamate–glutamine
translocator. The major pathway of nitrate assimilation is
printed bold.
nitrate transporters composed of seven members
in this species. Genes of both classes have been
found throughout the plant kingdom, including
C4 plants (Santi et al., 2003).
Nitrate is reduced to ammonium in two spatially
separated steps. In the cytosol, nitrate reductase
(NR) transfers electrons from NADH to nitrate to
form nitrite. Nitrite is transported into plastids and
reduced to ammonium by ferredoxin dependent
nitrite reductase. Ammonium is assimilated into
organic compounds by glutamine synthetase (GS)
which uses glutamate as the ammonium acceptor.
GS is coupled with glutamate synthase (GOGAT)
which transfers the amino group of glutamine to
2-oxoglutarate to form two molecules of gluta-
mate, in a GS/GOGAT cycle. The cycle thus uses
one molecule of 2-oxoglutarate to assimilate one
NH3 molecule and export one molecule of the
111
amino acid glutamate (Fig. 1, Miflin and Habash,
2002; Stitt et al., 2002; Weber and Flugge, 2002).
Glutamate is the source of reduced N for the syn-
thesis of other amino acids, with the first step
in this route being usually transamination with
oxaloacetate to form aspartate catalyzed by aspar-
tate aminotransferase.
Ammonium in plants originates not only from
nitrate reduction. It is present as a nutrient in
the soil and can be transported into plants by
ammonium transporters (von Wiren et al., 2000).
In fact, ammonium is the preferred source of
inorganic N for many plant species and optimal
growth is often achieved only when both nitrate
and ammonium are present (Bloom et al., 1993).
Ammonium is also produced by photorespira-
tion, in the mitochondrial glycine decarboxyla-
tion reaction (cf. Chapter 6, this volume; Linka
and Weber, 2005). An efficient assimilation and
re-assimilation of ammonium thus must occur in
all compartments to prevent its accumulation to
toxic levels. Indeed, plastidic and cytosolic iso-
forms of GS exist in all plant species (Inokuchi
et al., 2002) and the plastidic GS is dual targeted
also to mitochondria (Taira et al., 2004). GOGAT
is present in plants in multiple forms as well, the
major ferredoxin dependent enzyme is found
predominantly in leaves, whereas in non-photo-
synthetic cells NADH–GOGAT is the prevalent
isoform. Both forms of GOGAT are, however,
localized to plastids (Tobin and Yamaya, 2001).
Another enzyme, the glutamate dehydrogenase,
which in vitro synthesizes glutamate from ammo-
nium and 2-oxoglutarate, has been long impli-
cated to participate in ammonium assimilation.
Recent findings however suggest that the major
function of this enzyme is the provision of car-
bohydrate skeletons for carbon metabolism from
amino acids during protein degradation (Miflin
and Habash, 2002).
B. Regulation of Nitrate Assimilation
Nitrate and ammonium assimilation is strongly
regulated by the N demand of the plant and N
supply and is closely connected to carbon metab-
olism. The components of the pathway undergo
a coordinated regulation which often occurs on
multiple levels. So, nitrate uptake is induced in
the presence of nitrate and feedback inhibited
by amino acids and ammonium. It is regulated by
112
light and CO2 concentration via the availability
of carbohydrates (Rufty et al., 1989; Stitt et al.,
2002). Accordingly, nitrate uptake was demon-
strated to be under diurnal regulation with maxi-
mum activity during day and minimum activity at
night (Lejay et al., 1999). The decrease of uptake
at night can be reversed by feeding sucrose. The
coordination between N demand and N supply
in control of nitrate uptake was proposed to be
achieved by internal cycling of amino acids and/
or cytokinins between the roots and the shoots
(Gessler et al., 2004). Ammonium transport seems
to be regulated in a similar manner to nitrate
transport: it undergoes a day night rhythm and the
reduction of uptake in night can be prevented by
sucrose treatment and is inhibited by glutamine
(Lejay et al., 2003; Loque and von Wiren, 2004).
However, in contrast to nitrate transport ammo-
nium uptake seems to be regulated primarily by
local and not by systemic signals (Loque and von
Wiren, 2004).
NR is a well studied enzyme which undergoes
a complex regulation. NR expression is control-
led by circadian clock and it is also induced by
light and by sugars in the dark (Cheng et al., 1992;
Campbell, 1999). The level of NR transcript is
modulated by CO2 availability; it decreases when
CO2 assimilation is diminished, e.g. due to water
stress (Foyer et al., 1998) and increases upon
exposure of plants to elevated CO2 (Fonseca
et al., 1997). The rapid response of NR activity to
environmental stimuli is, however, caused mostly
by a rapid post-translational regulation. Upon
sudden decrease in photosynthesis NR is rapidly
and reversibly phosphorylated (Kaiser and Huber,
2001). The phosphorylation of NR enables its
binding to 14-3-3 proteins, which seems to be the
actual mechanism of inactivation and might be
a signal for NR degradation (Lillo et al., 2004).
The rapid response seems to be necessary to pre-
vent accumulation of toxic levels of nitrite. NR is
present in leaves and roots with different distribu-
tion of the activity among these organs in differ-
ent plant species. Even a relatively low activity,
however, is important for correct C/N balance
as revealed by experiments with tobacco lacking
root NR (Kruse et al., 2002).
A central role in controlling nitrate and ammo-
nium assimilation is occupied by GS. In contrast
to NR and nitrite reductase, which are usually
encoded by one to two genes, GS and GOGAT
Stanislav Kopriva
are encoded by small multigene families of three
to six members. This accounts for a great versa-
tility of regulation with some genes expressed in
tissue specific manner and at certain developmen-
tal stages while some being universal (Miflin and
Habash, 2002). The organel-targeted GS2 is espe-
cially important for reassimilation of the photores-
piratory NH3 and is, therefore, highly expressed
in green tissues and inducible by light (Oliveira
and Coruzzi, 1999). Cytosolic GS1 often fulfils
specialized roles, such as in nodules to assimilate
the ammonium produced by nitrogen fixation in
the bacteroids (Stanford et al., 1993) or in tissues
involved in transport of reduced N (Miflin and
Habash, 2002). GS is regulated transcription-
ally but undergoes also post-transcriptional and
post-translational regulation. Both GS forms are
subjected to phosphorylation and bind 14-3-3
proteins. Cytosolic GS is increased during
senescence to facilitate the remobilisation of N
(Habash et al., 2001). GS is also often associated
with improved yield of crop plants by quantita-
tive genetics (Hirel et al., 2001). Indeed, overex-
pression of pine cytosolic GS in poplar resulted
in significantly enhanced growth (Gallardo et al.,
1999) whereas disruptions of specific GS1 genes
in maize led to reduced kernel size and/or number
(Martin et al., 2006).
Despite the importance of N assimilation sur-
prisingly little is known about the molecular
mechanisms of the regulation of N acquisition and
metabolism. Nitrate transporter NRT1;1 seems
to play an important role in N sensing (Remans
et al., 2006). Nitrate has clearly a role as a signal
since microarray analysis of Arabidopsis mutants
in nitrate reductase revealed that expression of
595 genes including several transcription factors
responded to nitrate treatment (Wang et al., 2004).
Different amino acids act as feedback inhibitors
of N assimilation, whereas glutamine seems to be
most important in regulation of nitrate and ammo-
nium uptake, glutamate, cysteine, and asparagine
inhibit nitrate reduction (Stitt et al., 2002; Gessler
et al., 2004). Sugars are clearly involved in the
regulation of N assimilation, both as inductors
and repressors, when their content is low, however
the mechanisms of their action are not understood
(Stitt et al., 2002). A special role in regulation of
N assimilation, specifically in signalling N-status
of the plant, is played by the phytohormones
cytokinins. Cytokinins are produced in the root
7 Nitrogen and Sulfur in C4 Plants
at sufficient N supply and after transport into the
shoots induce expression of genes of N assimi-
lation (Wagner and Beck, 1993; Mok and Mok,
2001). They are, however, also transported from
the shoot to the root and affect nitrate uptake
(Collier et al., 2003).
C. Nitrate Assimilation in C4 Plants
Most of our knowledge on regulation of N metab-
olism has been derived from C3 plants and no dif-
ferences in organization and regulation of nitrate
assimilation between C3 and C4 plants were
described. However, C4 plants differ significantly
in the compartmentalization of N metabolism and
also in N use efficiency. It has long been known
that in terms of growth rate C4 grasses respond
better to applied N than C3 grasses (Hallock et al.,
1965). The C4 species clearly exhibited a higher N
use efficiency, expressed as biomass per unit N in
plant (Brown, 1978). The greater N use efficiency
seems to be connected with a lower content of
Rubisco in the leaves due to the CO2 concentrat-
ing mechanism. The N use efficiency in C4 plants
will be discussed in detail by Ghannoum et al. in
Chapter 8 of this volume.
The intracellular localization of N assimila-
tion in maize follows the pattern of C3 plants, i.e.
cytosolic localization of NR and the presence of
nitrite reductase in chloroplast (Ritenour et al.,
1966). Because of the observed differences in
distribution of carbon metabolism in C4 plants
and in structures of MS and BSC chloroplasts
the question of intercellular localization of nitrate
assimilation has frequently been addressed in
the early years of C4 photosynthesis research.
By enzyme activity assays Mellor and Tregunna
(1971) found that NR is prevalently present in
MC of three C4 species with different types of BSC
chloroplasts: maize, Sorghum sudanense, and
Gomphrena globosa. NADH dependent glutamate
dehydrogenase, which at that time was believed
to be the only ammonium assimilating enzyme,
was localized in BSC. Nitrite reductase activity
has been found in MC of maize and S. sudanense,
but in BSC of G. globosa which seems to imply
that it is associated with the presence of chloro-
plasts with grana (Mellor and Tregunna, 1971).
Also in Eleusine coracana NR and nitrite reduct-
ase occurred predominantly in MC (Rathnam
and Das, 1974). In a more systematic approach
113
analysing five C4 species of all three metabolic
subtypes, i.e. NADP-malic enzyme, NAD-malic
enzyme, and phoshoenolpyruvate carboxykinase,
NR, nitrite reductase, GS, and GOGAT were
coordinately localized in MC, except Panicum
maximum, where nitrite reductase was present
both in MC and BSC (Rathnam and Edwards,
1976). On the other hand, in another study with
maize, GS and GOGAT have been found in both
cell types but predominantly in BSC (Harel et al.,
1977). Similarly, in Digitaria sanguinalis NR
and nitrite reductase were found exclusively in
MC, GS was equally distributed between MC and
BSC, and GOGAT was predominantly localized
to BSC (Moore and Black, 1979).
Since all the localization studies were based on
distribution of enzyme activities in differentially
homogenized leaves, it was difficult to conclude
on exclusivity of the intercellular distribution of
the N assimilation pathway and its components.
Importantly, therefore, a clear evidence for an
exclusive localization of maize NR in the cytosol
of MC was obtained by immunogold labelling
(Vaughn and Campbell, 1988). Similarly, the
ambiguities in distribution of ammonium assimi-
lation were clarified by Becker et al. (1993) who
showed that both cytosolic and plastidic GS are
present in both cell types, whereas ferredoxin
dependent GOGAT is almost completely confined
to BSC chloroplasts.
An interesting feature of C4 plants is a rela-
tively high cytosolic GS activity. Whereas in most
C3 species cytosolic GS1 accounts for less than
30% of total leaf GS activity, in most C4 plants
analyzed to date GS1 activity is higher or equal
to the plastidic GS2 (McNally et al., 1983).
Analyzing Panicum species of different type of
photosynthesis Hirel et al. (1983) showed that the
presence of GS1 activity in leaf correlated with
C4 photosynthesis, with the C3–C4 intermediate
P. milliodes possessing foliar GS1 activity inter-
mediate between the C3 and C4 species. The ratio
in accumulation of the two GS isoforms also dif-
fers in between the two cell types of C4 plants.
GS1 seems to be more abundant in MC, whereas
both isoform are present at the same level in BSC
(Becker et al., 2000). Clearly, due to substantially
reduced and BSC confined photorespiration much
less ammonia is produced in C4 leaves than in
C3 leaves and therefore the demand for its rapid
refixation by GS is lower. The MC localization of
114 Stanislav Kopriva

nitrate reduction implies an increased need for
transport of reduced N between MC and BSC,
which might explain the need for higher cytosolic
GS. The evolutionary advantage of the spatial
distribution of nitrate assimilation in C4 plants
is, however, not known. Interestingly, assimila-
tion of another essential mineral nutrient, sulfate,
is also distributed in cell-specific manner in C4
plant species.
III. Sulfate Assimilation
Sulfur is essential for all living organisms as
a constituent of the amino acids cysteine and
methionine, many coenzymes such as iron sul-
fur centers, thiamine, lipoic acid, etc., and vari-
ous other compounds of primary and secondary
metabolism. In most of these compounds sulfur
is present in the reduced (−2) form of organic
sulfide. The most common form of sulfur in
nature is, however, oxidized as inorganic sulfate
(+6). Plants, algae, and many microorganisms
are able to directly utilize sulfate, reduce it and
incorporate into bioorganic molecules, which are
the form of sulfur accessible to animals and other
organisms. The pathway of sulfate assimilation
is thus an essential component of plant primary
metabolism.
acetylation of serine by serine acetyltransferase,
to form cysteine in a reaction catalyzed by O-ace-
tylserine (thiol)lyase (Fig. 2; Leustek et al., 2000;
Kopriva, 2006). Cysteine is the source of reduced
sulfur for synthesis of methionine, iron sulfur
clusters, and other compounds.
Sulfate assimilation is confined to plastids,
however, some reactions occur also in other com-
partments. The cysteine synthesis, e.g., proceeds
in all three compartments capable of protein syn-
thesis, i.e. plastids, cytosol, and mitochondria
(Wirtz et al., 2004). ATPS activity was detected
both in plastids and in the cytosol (Lunn et al.,
1990; Rotte and Leustek, 2000). On the other
hand the enzymes involved in reductive steps of
the pathway, APR and sulfite reductase, are local-
ized exclusively in plastids (Brunold and Suter,
1989; Prior et al., 1999; Koprivova et al., 2001).
Total foliar ATPS and APR activity decrease with

A. Plant Sulfate Assimilation

The pathway of sulfate assimilation in plants has

been completely resolved only relatively recently
(Suter et al., 2000) and has lately been subjected
to several comprehensive reviews (Leustek et al.,
2000; Saito, 2004; Rausch and Wachter, 2005;
Kopriva, 2006). Sulfate uptake into plant cells is
facilitated by sulfate transporters (Fig. 2). Also
sulfate transporters form a large multigene family
with 12–15 members differing in affinity to sul-
fate and tissue distribution (Buchner et al., 2004).
Because it is a very stable and inert compound,
before reduction sulfate has to be activated to Fig. 2. Schematic representation of plant sulfate assimila-
adenosine 5¢-phosphosulfate (APS) by adenyla- tion. Dark shaded rectangle represents mitochondria, light
tion catalyzed by ATP sulfurylase (ATPS). APS shaded one denotes plastid. Enzymes are symbolized by
is reduced to sulfite by APS reductase (APR). numbers: 1 – sulfate transporter, 2 – ATP sulfurylase, 3 –
Sulfite is further reduced to sulfide by ferre- APS reductase, 4 – sulfite reductase, 5 – serine acetyltrans-
ferase, 6 – O-acetylserine (thiol)lyase, 7 – g-glutamylcysteine
doxin dependent sulfite reductase. Sulfide is synthetase, 8 – glutathione synthetase, 9 – APS kinase, 10 –
than incorporated into the amino acid skeleton of sulfotransferase. The major pathway of sulfate assimilation
O-acetylserine (OAS), which is synthesized by and glutathione synthesis (in Arabidopsis) is printed bold.
7 Nitrogen and Sulfur in C4 Plants
the leaf age in Arabidopsis (von Arb and Brunold,
1986; Rotte and Leustek, 2000), however, the
cytosolic and plastidic ATPS are regulated dif-
ferently. Whereas the plastidic activity declines
with time, the cytosolic is increasing with leaf
age. This indicates different roles of ATPS in the
two compartments: a provision of APS for sulfate
reduction for biosynthetic processes required for
growth in plastids and involvement in synthesis
of secondary compounds in the cytosol (Rotte
and Leustek, 2000).
The reduction of sulfate occurs predominantly in
leaves, and reduced sulfur compounds are distrib-
uted to sink tissues via phloem (Herschbach and
Rennenberg, 2001). It appears, however, that most
tissues are capable of sulfate reduction, including
roots and developing seeds (Brunold and Suter,
1989; Tabe and Droux, 2002). Indeed, available
microarray data in the Genevestigator database
reveals the presence of mRNA for the genes of
sulfate assimilation, such as APR or sulfite reduct-
ase, in all Arabidopsis organs, including flowers
and siliques (Zimmermann et al., 2004). These
findings were corroborated using promoter::GUS
fusions which clearly showed activity of APR
and ATPS promoters in all tissues of Arabidopsis
(A Koprivova, C Matthewman, S Kopriva, 2008,
unpublished). However, whether the sulfate reduc-
tion rate in these cells is sufficient to cover their
needs for reduced sulfur instead of relying on long
distance transport of organic sulfur compounds,
such as glutathione or S-methylmethionine,
remains to be seen (Herschbach and Rennenberg,
2001). However, there is a group of plants that
lacks the ability to reduce sulfate in a great portion
of their cells, the monocot C4 plants (reviewed in
Kopriva and Koprivova, 2005).
B. Regulation of Sulfate Assimilation
The sulfate assimilation pathway is extensively
regulated in a demand-driven manner to prevent
accumulation of toxic intermediate and to provide
optimal rate of cysteine production (Lappartient
and Touraine, 1996; Leustek et al., 2000; Kopriva,
2006). Thus, when demand for reduced sulfur is
increased due to enhanced protein synthesis or
increased turnover of the sulfur containing tripep-
tide glutathione (see below) the flux through the
pathway is increased (Lappartient and Touraine,
1996). On the other hand, when plants are exposed
115
to reduced sulfur in the atmosphere or rhizosphere
or when there is not sufficient supply of the amino
acid acceptor due to carbon or nitrogen defi-
ciency, the pathway is downregulated (Koprivova
et al., 2000; Westerman et al., 2001; Kopriva et al.,
2002; Vauclare et al., 2002). Although regulation
of all components of the sulfate assimilation has
been described, control flux analysis revealed
that APR and sulfate uptake possess the highest
control over the pathway (Vauclare et al., 2002).
In line with these observations, APR activity and
mRNA accumulation undergoes a diurnal rhythm
with a maximum during light and minimum at
night (Kocsy et al., 1997; Kopriva et al., 1999).
The high activity coincides with the highest flux
through the pathway (Kopriva et al., 1999).
Despite a comprehensive knowledge on the
physiological responses of sulfate assimilation
to various environmental stimuli, very little is
known about the molecular mechanisms of reg-
ulation and the signals involved. Several com-
pounds have been proposed as molecular signals
in regulation of the pathway. OAS accumulates
during sulfur deficiency and when supplied exog-
enously induces mRNA accumulation of many
genes of sulfate uptake and assimilation (Neuen-
schwander et al., 1991; Koprivova et al., 2000;
Hopkins et al., 2005). Indeed, system biology
approaches revealed a correlation of OAS con-
tent with transcript accumulation of many genes
during a sulfur starvation response and large set
of genes regulated in the same way by sulfate
starvation and OAS treatment (Hirai et al., 2003,
2005). However, not all genes induced by sulfur
deficiency are regulated by OAS and the timing
of OAS accumulation seems to fall behind the
induction of sulfate uptake by sulfur starvation in
potato, so that OAS is probably not the only sig-
nal in the sulfur starvation response (Hirai et al.,
2003, 2005; Hopkins et al., 2005). Glutathione
(GSH) may also represent the signal of sulfur
status of the plant, as depletion of GSH by treat-
ment with an inhibitor of its synthesis, buthionine
sulfoximine (BSO), leads to upregulation of APR,
whereas GSH itself inhibits sulfate uptake and
reduction (Lappartient and Touraine, 1996; Vau-
clare et al., 2002; Hartmann et al., 2004). ATPS
and APR activity are reduced also in plants treated
with cysteine, however, the feedback inhibition is
alleviated when simultaneously BSO blocks the
synthesis of GSH from the additional cysteine
116
(Lappartient and Touraine, 1996; Vauclare et al.,
2002). In addition, several phytohormones have
been shown to modulate expression or activity
of various components of sulfate assimilation,
such as jasmonate (Harada et al., 2000; Jost et al.,
2005), cytokinins (Ohkama et al., 2002), or absci-
sic acid (Barroso et al., 1999). Only very recently,
however, the first transcription factor and cis ele-
ment responsible for regulating genes for sulfate
transporter by sulfur starvation have been identi-
fied (Maruyama-Nakashita et al., 2005, 2006).
C. Sulfate Assimilation in C4 Plants
In a search for further metabolic processes spa-
tially distributed in C4 plants it was soon discov-
ered that in maize 75–100% of total leaf ATPS
activity is confined to BSC (Gerwick and Black,
1979; Passera and Ghisi, 1982; Burnell, 1984;
Schmutz and Brunold, 1984). These findings
were extended to 17 other C4 species of all three
C4 subtypes, where 95–100% of total leaf ATPS
was localized in BSC chloroplasts (Gerwick
et al., 1980). Also APR was found almost exclu-
sively in BSC of maize (Schmutz and Brunold,
1984; Burgener et al., 1998), while the activities
of sulfite reductase and OAS(thiol)lyase could
be measured at comparable levels in MC and
BSC (Passera and Ghisi, 1982; Burnell, 1984;
Schmutz and Brunold, 1985). In attempts to deci-
pher the mechanism of the spatial distribution of
these enzymes, northern analysis of BSC and
MC specific RNA revealed that mRNA levels for
ATPS, APR, and sulfite reductase were detected
in BSC only, whereas the mRNA for OAS(thiol)
lyase was found in both MC and BSC (Kopriva
et al., 2001). The cell-specific localization of
enzymes of sulfate assimilation in maize seems,
therefore, to be regulated on the transcriptional
level, at least under standard growth conditions.
However, the situation might be different under
stress. Maize is especially sensitive to chilling
which induces a strong oxidative stress charac-
terized by production of reactive oxygen species
(ROS). In maize plants subjected to chilling APR
activity and mRNA level were greatly increased
in BSC, and mRNA but not enzyme activity was
also detectable in MC. This indicates an addi-
tional post-transcriptional mechanism to ensure the
BSC specific localization of sulfate assimilation in
maize (Kopriva et al., 2001).
Stanislav Kopriva
The exclusive localization of ATPS and APR
in BSC of maize means that an efficient transport
of reduced sulfur compounds from BSC to MC
must exist. MC are capable of cysteine synthe-
sis, therefore, the transport form of reduced sulfur
could be sulfide, cysteine, methionine or glutath-
ione. Feeding of isolated bundle sheath strands
from maize with [35S]sulfate resulted in secre-
tion of labelled cysteine into the nutrient solution
(Burgener et al., 1998). It seems therefore, that
cysteine (or its oxidized form cystine) is the most
probable transport metabolite for reduced sulfur
(Fig. 2). Interestingly, [35S]sulfate feeding experi-
ments also imply that glutathione synthesis was
predominantly localized in MC (Burgener et al.,
1998; and see below).
The biological significance of the BSC specific
localization of sulfate assimilation in C4 plants is
not obvious and, similarly, it is not clear whether
this localization is a pre-requisite or a consequence
of C4 photosynthesis. To answer this question we
addressed the distribution of ATPS and APR in
Flaveria species with different types of photosyn-
thesis (Koprivova et al., 2001). The dicot genus
Flaveria (Flaveriinae–Asteraceae) is an excellent
model to study the evolution of C4 photosynthesis
because, beside C3 and C4 species, it comprises
a relatively large number of C3–C4 intermediates
(Ku et al., 1991). A continuous gradation in the
physiology and biochemistry of C4 photosynthe-
sis can be found among Flaveria species (Monson
and Moore, 1989). Indeed, we showed previously
that the C3–C4 intermediate Flaveria species are
true evolutionary intermediates in the path from
C3 to C4 photosynthesis, based on phylogenetic
analysis of the H-protein subunit of glycine decar-
boxylase (Kopriva et al., 1996).
The aim of the study with Flaveria was to
compare the intercellular distribution of sulfate
assimilation in C3, C3–C4, and C4 species. We
expected a BSC localization of APR and ATPS in
C4 Flaveria species and a ubiquitous distribution
in C3 species. The distribution of the two enzymes
in the intermediate ones would be an excellent
indication for the evolutionary sequence of the
processes leading to the BSC localization of sul-
fate assimilation. Surprisingly however, northern
analysis of cell-specific RNA and in situ hybridi-
zation revealed that in the C4 species F. trinervia
mRNA for ATPS and APR were present at com-
parable levels in both MC and BSC. Immunogold
7 Nitrogen and Sulfur in C4 Plants
electron microscopy confirmed the presence of
APR protein in chloroplasts of both cell types
(Koprivova et al., 2001). Consequently, the local-
ization of assimilatory sulfate reduction in BSC
cannot be a general C4 trait.
How can these findings be explained taking
into account the results of Gerwick et al. (1980)
who showed the exclusive BSC localization of
ATPS in 17 C4 species? Whereas the 17 species
analyzed before were monocots, F. trinervia and
F. australasica were the first C4 dicots where
the subcellular localization of the pathway was
addressed. It has to be concluded that sulfate
assimilation is exclusively localized in BSC only
in C4 monocots and this distribution is thus nei-
ther a pre-requisite nor a consequence of C4 pho-
tosynthesis (Koprivova et al., 2001; Kopriva and
Koprivova, 2005). However, the BSC localization
of ATPS in maize and other C4 plants analyzed
by Gerwick et al. (1980) is not a general trait of
monocots either, since in wheat, a C3 monocot,
ATPS and APR are present in all cell types (Sch-
mutz and Brunold, 1984). Whether C4 Flaveria
species are exceptions or the rule for C4 dicots
remains to be established, however, it is evi-
dent that the previously generally accepted link
between C4 photosynthesis and BSC localization
of sulfate assimilation is no longer valid.
The investigations of sulfate assimilation in
Flaveria resulted in an additional interesting
finding. The APR activity and levels of thiols
were significantly higher in leaves of C4-like and
C4 species than in those of C3 and C3–C4 spe-
cies (Koprivova et al., 2001). APR, cysteine and
GSH content correlated with the degree of devel-
opment of C4 photosynthesis expressed by CO2
compensation points (Kopriva and Koprivova,
2005). The actual foliar concentration of GSH is
highly dependent on environmental conditions
and, therefore, varies significantly among differ-
ent plant species but also within single species.
Since the Flaveria species were grown at identical
controlled conditions, it seems that the clear ten-
dency to towards higher APR activity and GSH
content with increasing C4 photosynthesis might
be a result of the adaptation to different habitats.
C4 photosynthesis is especially advantageous in
dry and warm conditions. The higher GSH con-
tents in C4 Flaveria might thus be a mechanism
to cope with increased oxidative stress caused by
such environmental conditions. According to the
117
demand-driven regulation, APR activity would
have to be elevated to supply sufficient cysteine
for the GSH synthesis.
IV. Glutathione Synthesis and Reduction
Among sulfur containing metabolites the tripep-
tide glutathione (g-glutamyl-cysteinyl-glycine)
plays the most versatile role being essential for
plant stress defense, redox regulation and signal-
ing, control of cell cycle, and as a storage and
transport form of reduced sulfur (May et al.,
1998a; Noctor et al., 1998a; Foyer and Rennen-
berg, 2000). GSH is involved in plant defense
against ROS as a reductant of dehydroascorbate
in the glutathione–ascorbate cycle (Noctor and
Foyer, 1998). It is indispensable for protection
against heavy metals as a substrate for synthe-
sis of phytochelatins, which chelate the metals
and enable their sequestration into the vacuoles
(Cobbett and Goldsbrough, 2002). Conjugation
of xenobiotics with GSH is the first step in their
detoxification and a molecular basis of resist-
ance to some herbicides (Dixon et al., 1998). Not
surprisingly, GSH accumulates to high levels
reaching concentration of several mM (Meyer
and Fricker, 2000; Hartmann et al., 2003). Due to
its role in stress defense and specifically in the
response to chilling, GSH synthesis and its regu-
lation was often addressed in C4 plants (Ruegseg-
ger and Brunold, 1993; Doulis et al., 1997; Kocsy
et al., 2001; Gómez et al., 2004; Kopriva and
Koprivova, 2005).
A. Regulation of GSH Synthesis
GSH is synthesized from its constituent amino
acids in two ATP dependent steps. Firstly a
g-glutamylcysteine synthetase (g-ECS) synthe-
sizes g-glutamylcysteine (g-EC) from glutamate
and cysteine. Subsequently, glycine is added to
the g-EC by glutathione synthetase (GSHS). GSH
synthesis is an essential process, since disruption
of gECS gene by T-DNA insertion is embryo-
lethal (Cairns et al., 2006). The rate of GSH syn-
thesis is primarily controlled by the availability
of the constituent amino acids, with the regula-
tion of g-ECS playing an additional substantial
role (Kopriva and Rennenberg, 2004). Most of
our knowledge on this regulation is derived from
118
studies with poplar overexpressing bacterial genes
for g-ECS and GSHS (e.g. Strohm et al., 1995;
Noctor et al., 1996, 1998b). g-ECS and GSHS are
induced at conditions of high demand for GSH,
such as after exposure to heavy metals or herbi-
cide safeners (Ruegsegger and Brunold, 1992;
Farago and Brunold, 1994; Schaefer et al., 1998).
g-ECS is more important for the control of GSH
synthesis because GSH content was increased
only in poplars overexpressing g-ECS and not
GSHS (Strohm et al., 1995). The enzyme seems
to undergo a complex regulation on different lev-
els. Heavy metals induce mRNA accumulation of
g-ECS (and GSHS) (Schaefer et al., 1998; Xiang
and Oliver, 1998), but a post-transcriptional regu-
lation has also been described (May et al., 1998b).
On the other hand, the enzyme is feedback inhib-
ited by GSH, which causes a reversible confor-
mational change due to a reduction of an internal
disulfide bond (Jez et al., 2004; Hothorn et al.,
2006). The g-ECS regulation thus seems to follow
the same demand-driven manner as described for
the components of sulfate assimilation. Indeed,
since at most physiological situations cysteine
limits GSH synthesis gECS is often regulated in
the same way as APR or other enzymes of sulfate
assimilation (Ruegsegger et al., 1990; Brunner
et al., 1995). At some conditions however, e.g.
during night or at non-photorespiratory condi-
tions, synthesis of GSH is controlled by availability
of glycine (Noctor et al., 1999).
During its function in the glutathione–ascorbate
cycle GSH is oxidized. The oxidized form of
glutathione, GSSG, is reduced by the action of
glutathione reductase (GR) which utilizes the
electrons from NADPH. GR maintains the reduc-
ing environment in cells so that GSSG normally
forms no more than 5% of total glutathione.
Increased ratio of GSSG to total GSH is thus
indicative of oxidative stress (Mullineaux and
Rausch, 2005).
B. Localization of GSH and GSH
Synthesis
GSH is present in all cellular compartments; how-
ever, the quantitative distribution is not resolved
yet. Currently, two approaches are being used to
quantify GSH on the subcellular level. The con-
focal laser scanning microscopy method makes
use of the fluorescence of the GSH conjugate
Stanislav Kopriva
with monochlorobimane (Meyer et al., 2001).
However, since the conjugation is catalyzed enzy-
matically by GSH transferase, the method can
directly measure only cytosolic GSH, whereas
the organellar pools can be estimated by HPLC
after cell disruption and additional chemical
labeling of the remaining GSH by monobro-
mobimane (Hartmann et al., 2003). The second
method is based on immunohistochemistry with
antibodies against GSH (Zechmann et al., 2005).
This approach shows the highest density of labe-
ling in mitochondria and the lowest in plastids.
Unfortunately, none of the methods have been
used for localization of GSH in C4 plants.
GSH synthesis has been shown to take place
in the cytosol and plastids (Hell and Bergmann,
1990; Noctor et al., 1998a). However, recently it
was revealed that the two steps of GSH biosyn-
thesis may be spatially separated, at least in some
plant species. In Arabidopsis and Brassica juncea
g-ECS seems to be exclusively localized in the
plastids whereas GSHS is present in both plas-
tids and cytosol (Wachter et al., 2005). The g-EC
may thus not only be a precursor of GSH but also
play important roles in transport of reduced sulfur
from plastids to the cytosol and possibly in sign-
aling of the redox status of the chloroplast. This
conclusion is supported by the identification of
the regulator of APX2 (rax1) mutant in Arabidop-
sis (Ball et al., 2004). This mutant constitutively
expresses cytosolic ascorbate peroxidase, which
is normally inducible by photooxidative stress,
and contains only approximately 50% of normal
foliar GSH levels. In rax1 thus the chloroplast–
cytosol signaling is clearly disturbed. The rax1
phenotype is caused by an R(229)-K substitution
in g-ECS protein (Ball et al., 2004). Several other
Arabidopsis mutants are associated with g-ECS
and low GSH levels, the cadmium sensitive cad2
(Cobbett et al., 1998), root meristemless rml1
(Vernoux et al., 2000), and phytoalexin-deficient
pad2 (Parisy et al., 2007), but none of them dis-
plays the same effect on the chloroplast–cytosol
signaling.
On the other hand, g-ECS and GSHS were
detected by immunolocalization in both chloro-
plasts and cytosol of maize (Gómez et al., 2004).
Also, expression of the bacterial gene for g-ECS
both in plastids and in the cytosol led to an
increase in foliar GSH content in poplar (Noctor
et al., 1996, 1998b). Interestingly, the increase
7 Nitrogen and Sulfur in C4 Plants
in GSH content in poplar plants overexpressing
g-ECS in the cytosol seems to be confined to this
compartment which implies a limited exchange
of GSH between cytosol and plastids (Hartmann
et al., 2003). GR is localized in cytosol, plastids,
and mitochondria (Edwards et al., 1990). Inter-
estingly, a single gene encodes both plastidic and
mitochondrial isoform of GR due to a presence
of a dual-specificity targeting peptide (Chew
et al., 2003).
GSH is present and can be synthesized in all
plant organs. Obviously, in many occasions cell
specific differences in GSH levels have been
found. In maize roots, GSH levels form a clear
gradient from the highest at root tip to the lowest
in the mature portion of the root (Kopriva et al.,
2001). When investigated at the cellular level
by confocal laser scanning microscopy approxi-
mately twofold higher cytosolic GSH concen-
tration was measured in atrichoblasts than in
trichoblasts (Meyer and Fricker, 2000). In addition
high GSH levels were detected in root cap while
markedly lower GSH was found in quiescent
centers (Sanchez-Fernandez et al., 1997). On the
other hand, in poplar leaves analyzed by the same
approach surprisingly uniform concentration of
cytosolic GSH has been detected (Hartmann
et al., 2003). Also in leaves, however, certain cell
types distinctly differ, as very high GSH concen-
tration has been found in leaf trichoms (Gutierrez-
Alcala et al., 2000). Consequently, gECS, GSHS,
and genes for enzymes of cysteine synthesis were
highly expressed in these cells. These cell specific
differences in GSH levels are probably caused by
differential rates of GSH synthesis and indicate
that GSH transport between cells may not be effi-
cient enough to balance such differences.
C. GSH Synthesis in C4 Plants
Due to its role in detoxification of ROS GSH is
particularly important in low temperature sensitive
C4 plants, such as maize, since chilling induces
oxidative stress via the photochemical production
of H2O2. Consequently, at low temperatures GSH
content and reduction state are higher in chilling
tolerant genotypes of tomato, Sorghum, wheat,
and maize than in the sensitive ones (Walker and
McKersie, 1993; Kocsy et al., 1996, 2000a; Badiani
et al., 1993; see Chapter 10, this volume). Brunner
et al. (1995) demonstrated that in maize chilling
119
caused the content of GSH to increase and, in
accordance with the demand-driven model of
regulation, also enzyme activities of APR, g-ECS,
and GSHS. Similarly, at 12°C the activities of APR
and GR and GSH content were higher in a chill-
ing tolerant maize genotype than in a sensitive one
(Kocsy et al., 1997). Accordingly, treatment with
1 mM BSO, which decreased GSH content to very
low levels, resulted in reduction of fresh weight
and in visible leaf injury of chilling-tolerant maize
at 5°C but not at ambient temperature (Kocsy
et al., 2000b). Addition of GSH or g-EC together
with BSO protected the plants from the chilling
injury by increasing GSH content and GR activity.
Similarly, when GSH content in the chilling sensi-
tive maize had been increased via treatment with
herbicide safeners, the chilling induced injury
was significantly reduced (Kocsy et al., 2001).
A simultaneous addition of BSO counteracted the
safener-induced protection. These experiments
thus clearly showed that, at least in maize, sen-
sitivity to chilling is a trait connected with GSH
content and/or reduction state (see Chapter 10, this
volume).
Despite its essential function in stress defense
GSH is not equally distributed between MC and
BSC in maize. GSHS activity is greater in MC
than in BSC resulting in a predominant GSH syn-
thesis rate in the MC (Burgener et al., 1998) and
higher GSH levels in this cell type (Doulis et al.,
1997; Burgener et al., 1998; Kopriva et al., 2001).
Surprisingly, GR was found exclusively in MC
of maize (Doulis et al., 1997; Pastori et al., 2000).
The MC specific localization of GR might be
explained by a limited capacity for NADPH pro-
duction in BSC. GR mRNA, however, was found
in both cell types revealing involvement of a
post-transcriptional regulation of its cell-specific
distribution (Pastori et al., 2000). In contrast to
GR, the enzymes of GSH synthesis were local-
ized in both MC and BSC of maize by immuno-
histochemistry (Gómez et al., 2004). In line with
previous findings foliar GSH content increased
in cold treated plants, however, chilling caused
induction of g-ECS mRNA in BSC but not in MC
(Gómez et al., 2004). It seems therefore, that both
cell types possess the capacity to synthesize GSH,
but the enzymes in BSC are more affected by
stress. The apparent contrast of these results with
previous data (Doulis et al., 1997; Burgener et al.,
1998) is likely to be caused by cell-type specific
120
post-translational modification of the enzymes
resulting in changes in GSH synthesis rates and
thus distribution of GSH. As g-ECS is redox reg-
ulated, variation in redox environment in MC and
BSC can be responsible for such differences in
activity. The preferential stress response in BSC
might be explained by the fact that cysteine, the
limiting factor in GSH synthesis, is synthesized
only in BSC and can be used for GSH synthe-
sis without the necessity for any transport steps
(Burgener et al., 1998; Kopriva et al., 2001). On
the other hand, the oxidized form of glutathione
can probably be reduced only in MC (Pastori
et al., 2000). Consequently, GSSG formed during
the stress in BSC has to be transported to MC for
reduction and thus the GSH pool in MC increases
while the BSC pool becomes depleted. This is
likely to result in increased demand for GSH
synthesis in BSC but not in MC and in activation
g-ECS and sulfate assimilation.
V. Physiological Significance
of the Distribution of Nitrate and Sulfate
Assimilation
Nitrate and sulfate assimilation are clearly local-
ized in MC and BSC, respectively, in many C4
plants. For sulfate assimilation it is obvious that
this localization is not linked to C4 photosynthe-
sis, as C4 Flaveria species possess the pathway
in both cell types. The physiological significance
of this localization and evolutionary advantage is,
however, not evident as yet.
A. Open Questions on Nitrate
Assimilation in C4 Plants
The cell specific distribution of N assimilation
in C4 plants is well established, however, many
questions are still open. Obviously, our findings
on sulfate assimilation in Flaveria (Koprivova
et al., 2001) incite questioning the universality
of results on N assimilation derived from maize
and a few other C4 species. Although similarly to
Gerwick et al. (1980) C4 species of all three types
were analyzed for localization of NR and GS with
the same result, no dicot species were included
(Rathnam and Edwards, 1976; McNally et al.,
1983). Only in the very first analysis by Mellor
and Tregunna (1971) a C4 dicot (G. globosa) was
Stanislav Kopriva
analyzed and, indeed, the results pointed to pos-
sible alternative distribution of the enzymes in
this species compared, e.g. to maize. The findings
have to be interpreted with caution, obviously,
due to the methodology based on enzyme activi-
ties in MC and BSC specific extracts obtained by
differential tissue extraction, which surely have
been cross-contaminated. Nevertheless, unlike in
other species analyzed, nitrite reductase activity
in G. globosa was higher in BSC than in MC.
In analogy with sulfate assimilation, this would
imply that the MC distribution of nitrate assimi-
lation may not be universal and not be connected
with C4 photosynthetic mechanism.
A second question concerns the increased N
use efficiency of C4 plants. Reduced accumula-
tion of Rubisco, the major sink for reduced N
in C3 plants, is thought to be the major reason for
better N use efficiency, as less N is required for the
same or a better CO2 fixation rate (Brown, 1978).
Whether the cell-specific distribution of NR and/
or the high content of cytosolic GS contribute to
the improved N use efficiency is not clear.
The physiological consequences of the altera-
tions in localization of N assimilation are also not
known. Major differences in regulation of N uptake
and assimilation between C3 and C4 plants have
not been reported. Clearly, more transport steps
are necessary to provide all cells with sufficient
reduced N and the MC with nitrate (Fig. 3). On the
other hand, the MC localization of NR and nitrite
reductase might prevent competition for reduction
equivalents between nitrate and CO2 assimilation
(Moore and Black, 1979). Alternatively, the low
NADPH production capacity in BSC chloroplasts
due to the lack of grana and photosystem II was
cited as a reason for MC localization of NR (Mel-
lor and Tregunna, 1971). This may be true for
maize and some other NADP-malic enzyme-type
C4 plants, but plants of the other types do possess
photosystem II in BSC and still reduce nitrate in
MC only (Rathnam and Edwards, 1976; Ketchner
and Sayre, 1992). Most likely explanation takes
into account that nitrite is an alternative accep-
tor of photosynthetic electrons and its reduction
is coupled to oxygen evolution (Miflin, 1972).
Localization of this reaction in MC chloroplast
thus prevents O2 evolution in BSC, which would
counteract the CO2 concentrating mechanism of
C4 plants and support the oxygenase reaction
of Rubisco (Moore and Black, 1979). Limitation
7 Nitrogen and Sulfur in C4 Plants
Fig. 3. Schematic representation of distribution of major steps in assimilation of carbon, nitrogen, and sulfur between mesophyll
(MC) and bundle sheath (BSC) of maize. Transport steps of C compounds are marked by dotted arrows, dashed and full arrows
symbolize transport of N and S compounds, respectively (Reprinted from Kopriva and Koprivova, 2005).
of NR to MC is essential to prevent accumulation
of nitrite and its toxicity in BSC. This might be
the biggest evolutionary advantage of the cell-
specific distribution of N assimilation in C4 plants.
This explanation would be strengthened if the MC
localization of NR and nitrite reductase would be
confirmed also for the dicot C4 species.
B. Significance of BSC Localization
of Sulfate Assimilation
Although not necessarily linked with C4 photo-
synthetic mechanism, sulfate is reduced only in
BSC of maize. A link with low rate of NADPH
production as discussed for nitrate assimilation
and GSH reduction (Mellor and Tregunna, 1971;
Doulis et al., 1997) can be excluded since then the
sulfate reduction would have to be localized in
MC. Burgener et al. (1998) speculated that low
concentration of oxygen in BSC would prevent
oxidation of intermediates of sulfate assimila-
tion, sulfite and sulfide, and thus increase the
efficiency of the pathway. However, if such oxi-
dation would indeed significantly reduce the rate
of sulfate assimilation, the pathway would not be
functional in chloroplasts of C3 plants. Conse-
quently, sulfate would be reduced in mitochon-
dria, what actually is the case in Euglena gracilis
(Brunold and Schiff, 1976), or elaborate structures
121
would have evolved possibly including bacterial
symbiosis, such as for N2 fixation in legumes.
Another attractive explanation is the
co-localization with photorespiration, namely
with GDC, the major source of serine in plants
since activated serine is the acceptor of sulfide
and a direct precursor of cysteine. However, serine
synthesized in BSC must be transported into MC
for protein synthesis anyway and thus a shortage
of this amino acid in MC seems unlikely. Moreo-
ver, in C4 Flaveria species GDC is BSC specific
but sulfate assimilation is not (Hylton et al., 1988;
Koprivova et al., 2001). In addition, the MC spe-
cific ferredoxin FdI (Matsumura et al., 1999) was
an efficient electron donor for sulfite reductase
(Yonekura-Sakakibara et al., 2000), so cell specific
differences in electron flux can also be excluded.
To find out the significance of the BSC specific
distribution of sulfate assimilation, one should
ask for the advantage maize may have gained this
way. Unfortunately, there does not seem to be any
obvious one. In contrast to nitrogen nutrition, a
difference in sulfur use efficiency between C3 and
C4 plants was not observed. As with other meta-
bolic processes, maize probably invests less into
synthesis of the proteins of sulfate assimilation
pathway, but on the other hand it must possess an
efficient transport system for cysteine and GSH.
Maize does not require significantly more or
122
less sulfate than other plant species and it is not
known for especially good or poor sulfur use effi-
ciency. It is not particularly resistant or sensitive
to heavy metals, which trigger a high demand for
reduced sulfur (Nussbaum et al., 1988). On the
other hand, sulfate assimilation and GSH synthe-
sis are associated with tolerance to chilling and
detoxification of herbicides, so the BSC localiza-
tion might even be a limitation for the capacity to
provide sufficient GSH to cope with such stress.
It seems, therefore, that the significance of com-
partmentalization of sulfate assimilation in maize
will further remain an open question.
C. Consequences of BSC Localization
of Sulfate Assimilation
To understand the cell-specific localization of
sulfate assimilation one also has to ask about its
consequences. Maize was a frequent subject of
investigations of assimilatory sulfate reduction in
the pre-Arabidopsis era. The results on regulation
of sulfate assimilation obtained with maize fitted
well to the general hypothesis of demand driven
control (Lappartient and Touraine, 1996). Coor-
dinate increase in mRNA levels for sulfate trans-
porters, ATPS, and APR was observed in maize
roots and leaves upon sulfate starvation (Bolchi
et al., 1999; Hopkins et al., 2004) and the ATPS
mRNA level was repressed in presence of reduced
sulfur compounds (Bolchi et al., 1999). Accord-
ingly, ATPS and APR activities were increased
upon treatments of maize with cadmium or chill-
ing (Brunner et al., 1995; Nussbaum et al., 1988;
Ruegsegger and Brunold, 1992). In all these
reports the regulation of sulfate assimilation in
maize was not distinguishable from other plants,
such as Lemna minor, poplar, potato, and Ara-
bidopsis (Kopriva, 2006). Differences appeared,
however, when the mechanisms of regulation
were addressed (Bolchi et al., 1999). To identify
the signal responsible for feedback inhibition of
sulfate assimilation by thiols, plants can be treated
with cysteine, glutathione, and cysteine together
with BSO which prevents its conversion to GSH.
Such analysis performed with Brassica napus,
Arabidopsis, and poplar unambiguously identi-
fied GSH as the molecular regulator (Lappar-
tient and Touraine, 1996; Vauclare et al., 2002;
Hartmann et al. 2004) whereas in maize cysteine
acted directly without the necessity of conversion
Stanislav Kopriva
to GSH (Bolchi et al., 1999). The molecular
mechanisms of this feedback inhibition are not
known, but it is reasonable to expect that the
responsible sensor/transcription factor in maize
is specific for cysteine whereas the correspond-
ing protein(s) in other plants are GSH specific.
This variation might well be a consequence of the
BSC localization of sulfate assimilation. As GSH
can be synthesized both in MC and BSC (Gómez
et al., 2004) but only cysteine is transported out of
BSC protoplasts (Burgener et al., 1998) it is likely
that the GSH pools in MC and BSC are not rapidly
interchangeable. On the other hand, cysteine pools
in the two cell types have to be linked to enable
efficient protein and GSH synthesis in MC and
rapid modulation of cysteine biosynthesis in BSC
upon even subtle changes in demand for reduced
sulfur in the whole leaf. Therefore, cysteine may
be much better suited as a signal of the sulfur
status at the site of sulfate assimilation than GSH.
VI. Conclusions
Nitrate and sulfate assimilation in C4 plants
are another processes differentially distributed
between MC and BSC. MC specific localization
of nitrate assimilation has been demonstrated in
only a few species and despite being generally
accepted, it has to be proven that this indeed is
a general C4 trait. This is even more imperative
after it was revealed that the BSC association of
sulfate assimilation is not a general feature of
C4 photosynthesis but is probably limited to C4
monocots. For both pathways the evolutionary
advantage of such compartmentalization is not
understood and should be addressed in future stud-
ies. Given the high interest in improvements of
nutrient use efficiency of crops the N metabolism
in C4 plants should certainly be further explored
to find whether the C4 specific alterations contrib-
ute to the improved N use efficiency of C4 plants.
Clearly, there are still more open questions than
answers in N and S metabolism of C4 plants.
Acknowledgments
Research in Stanislav Kopriva’s laboratory at John
Innes Centre is supported by the Biotechnology and
Biological Sciences Research Council (BBSRC).
7 Nitrogen and Sulfur in C4 Plants
References
Badiani M, Paolacci AR, D’Annibale A and Sermanni GG
(1993) Antioxidants and photosynthesis in the leaves of
Triticum durum L. seedlings acclimated to low, non-chilling
temperature. J Plant Physiol 142: 18–24
Ball L, Accotto GP, Bechtold U, Creissen G, Funck D,
Jimenez A, Kular B, Leyland N, Mejia-Carranza J, Reynolds
H, Karpinski S and Mullineaux PM (2004) Evidence for
direct link between glutathione biosynthesis and stress
defense gene expression in Arabidopsis. Plant Cell 16:
2446–2462
Barroso C, Romero LC, Cejudo FJ, Vega JM and Gotor
C (1999) Salt-specific regulation of the cytosolic
O-acetylserine(thiol)lyase gene from Arabidopsis thal-
iana is dependent on abscisic acid. Plant Mol Biol 40:
729–736
Bassüner B, Keerberg O, Bauwe H, Pyarnik T and Keer-
berg H (1984) Photosynthetic CO2 metabolism in C3-C4
intermediate and C4 species of Flaveria (Asteraceae).
Biochem Physiol Pflanzen 179: 631–634
Bauwe H (1984) Photosynthetic enzyme activities and
immunofluorescence studies on the localization of ribu-
lose-1,5-bisphosphate carboxylase/oxygenase in leaves of
C3, C4, and C3-C4 intermediate species of Flaveria (Aster-
aceae). Biochem Phys Pflanzen 179: 253–268
Becker TW, Perrot-Rechenmann C, Suzuki A and Hirel B
(1993) Subcellular and immunocytochemical localiza-
tion of the enzymes involved in ammonia assimilation in
mesophyll and bundle-sheath cells of maize leaves. Planta
191: 129–136
Becker TW, Carrayol E and Hirel B (2000) Glutamine syn-
thetase and glutamate dehydrogenase isoforms in maize
leaves: localization, relative proportion and their role in
ammonium assimilation or nitrogen transport. Planta 211:
800–806
Bloom AJ, Jackson LE and Smart DR (1993) Root growth as
a function of ammonium and nitrate in the root zone. Plant
Cell Environ 16: 1294–1301
Bolchi A, Petrucco S, Tenca PL, Foroni C and Ottonello S
(1999) Coordinate modulation of maize sulfate permease
and ATP sulfurylase mRNAs in response to variations in
sulfur nutritional status: stereospecific down-regulation
by L-cysteine. Plant Mol Biol 39: 527–537
Brown RH (1978) A difference in N use efficiency in C3 and
C4 plants and its implications in adaptation and evolution.
Crop Sci 18: 93–98
Brunner M, Kocsy G, Rüegsegger A, Schmutz D and Brunold
C (1995) Effect of chilling on assimilatory sulfate reduc-
tion and glutathione synthesis in maize. J Plant Physiol
146: 743–747
Brunold C and Schiff JA (1976) Studies of sulfate utilization
by algae. 15. Enzymes of assimilatory sulfate reduction
in Euglena and their cellular localization. Plant Physiol
57: 430–436
123
Brunold C and Suter M (1989) Localization of enzymes of
assimilatory sulfate reduction in pea roots. Planta 179:
228–234
Buchner P, Takahashi H and Hawkesford MJ (2004) Plant
sulphate transporters: co-ordination of uptake, intracellu-
lar and long-distance transport. J Exp Bot 55: 1765–1773
Burgener M, Suter M, Jones S and Brunold C (1998) Cyst(e)
ine is the transport metabolite of assimilated sulfur from
bundle-sheath to mesophyll cells in maize leaves. Plant
Physiol 116: 1315–1322
Burnell JN (1984) Sulfate assimilation in C4 plants. Plant
Physiol 75: 873–875
Cairns NG, Pasternak M, Wachter A, Cobbett CS and Meyer
AJ (2006) Maturation of Arabidopsis seeds is depend-
ent on glutathione biosynthesis within the embryo. Plant
Physiol 141: 446–455
Chalot M and Brun A (1998) Physiology of organic nitro-
gen acquisition by ectomycorrhizal fungi and ectomycor-
rhizas. FEMS Microbiol Lett 22: 21–44
Campbell WH (1999) Nitrate reductase structure, function
and regulation: bridging the gap between biochemistry
and physiology. Annu Rev Plant Physiol Plant Mol Biol
50: 277–303
Cheng CL, Acedo GN, Cristinsin M and Conkling MA
(1992) Sucrose mimics the light induction of Arabidopsis
nitrate reductase gene transcription. Proc Natl Acad Sci
USA 89: 1861–1864
Chew O, Whelan J and Millar AH (2003) Molecular defi-
nition of the ascorbate-glutathione cycle in Arabidop-
sis mitochondria reveals dual targeting of antioxidant
defenses in plants. J Biol Chem 278: 46869–46877
Cobbett C and Goldsbrough P (2002) Phytochelatins and
metallothioneins: roles in heavy metal detoxification and
homeostasis. Annu Rev Plant Biol 53: 159–182
Cobbett CS, May MJ, Howden R and Rolls B (1998)
The glutathione-deficient, cadmium-sensitive mutant,
cad2-1, of Arabidopsis thaliana is deficient in gamma-
glutamylcysteine synthetase. Plant J 16: 73–78
Collier M, Fotelli M, Nahm M, Kopriva S, Rennenberg H,
Hanke D and Geßler A (2003) Regulation of nitrogen
uptake by Fagus sylvatica on a whole plant level- Interac-
tions between cytokinins and soluble N compounds. Plant
Cell Environ 26: 1549–1560
Day DA, Poole PS, Tyerman SD and Rosendahl L (2001)
Ammonia and amino acid transport across symbiotic
membranes in nitrogen-fixing legume nodules. Cell Mol
Life Sci 58: 61–71
Dixon DP, Cummins L, Cole DJ and Edwards R (1998)
Glutathione-mediated detoxification systems in plants.
Curr Opin Plant Biol 1: 258–266
Doulis AG, Debian N, Kingston-Smith AH and Foyer CH
(1997) Differential localization of antioxidants in maize
leaves. Plant Physiol 114: 1031–1037
Edwards E, Rawsthorne S and Mullineaux P (1990) Sub-
cellular distribution of multiple forms of glutathione
124
reductase in leaves of pea (Pisum sativum L.). Planta 180:
278–284
Edwards GE, Franceschi VR, Ku MS, Voznesenskaya EV,
Pyankov VI and Andreo CS (2001) Compartmentation of
photosynthesis in cells and tissues of C4 plants. J Exp Bot
52: 577–590
Farago S and Brunold C (1994) Regulation of thiol contents
in maize roots by intermediates and effectors of glutath-
ione synthesis. J Plant Physiol 144: 433–437
Fonseca F, Bowsher CG and Stulen I (1997) Impact of
elevated atmospheric CO2 on nitrate reductase transcrip-
tion and activity in leaves and roots of Plantago major.
Physiol Plant 100: 940–948
Foyer CH and Rennenberg H (2000) Regulation of glutathione
synthesis and its role in abiotic and biotic stress defence. In:
Brunold C et al (eds) Sulfur nutrition and sulfur assimilation
in higher plants: molecular, biochemical and physiological
aspects, pp 127–153. Paul Haupt, Bern, Switzerland
Foyer CH, Valadier MH, Migge A and Becker TW (1998)
Drought-induced effects on nitrate reductase activity and
mRNA and on the coordination of nitrogen and carbon
metabolism in maize leaves. Plant Physiol 117: 283–292
Gallardo F, Fu J, Canton FR, Garcia-Gutierrez A, Cano-
vas FM and Kirby EG (1999) Expression of a conifer
glutamine synthetase gene in transgenic poplar. Planta
210: 19–26
Gerwick BC and Black CC (1979) Sulfur assimilation in C4
plants. Plant Physiol 64: 590–593
Gerwick BC, Ku SB and Black CC (1980) Initiation of sul-
fate activation: a variation in C4 photosynthesis plants.
Science 209: 513–515
Gessler A, Kopriva S and Rennenberg H (2004) Regula-
tion of nitrate uptake at the whole-tree level: interaction
between nitrogen compounds, cytokinins and carbon
metabolism. Tree Physiol 24: 1313–1321
Gómez LD, Vanacker H, Buchner P, Noctor G and Foyer CH
(2004) Intercellular distribution of glutathione synthesis
in maize leaves and its response to short-term chilling.
Plant Physiol 134: 1662–1671
Gutierrez-Alcala G, Gotor C, Meyer AJ, Fricker M, Vega
JM and Romero LC (2000) Glutathione biosynthesis in
Arabidopsis trichome cells. Proc Natl Acad Sci USA 97:
11108–11113
Habash DZ, Massiah AJ, Rong HL, Wallsgrove RM and
Leigh RA (2001) The role of cytosolic glutamine syn-
thetase in wheat. Ann Apl Biol 138: 83–89
Hallock DL, Brown RH and Blaser RE (1965) Relative yield
and composition of Kentucky 31 fescue and coastal ber-
mudagrass at four nitrogen levels. Agron J 57: 539–542
Harada E, Kusano T and Sano H (2000) Differential expres-
sion of genes encoding enzymes involved in sulfur assim-
ilation pathways in response to wounding and jasmonate
in Arabidopsis thaliana. J Plant Physiol 156: 272–276
Harel E, Lea PJ, and Miflin BJ (1977) The localisation of
enzymes of nitrogen assimilation in maize leaves and
their activities during greening. Planta 134: 195–200
Stanislav Kopriva
Hartmann TN, Fricker MD, Rennenberg H and Meyer AJ
(2003) Cell-specific measurement of cytosolic glutath-
ione in poplar leaves. Plant Cell Environ 26: 965–975
Hartmann T, Hönicke P, Wirtz M, Hell R, Rennenberg H and
Kopriva S (2004) Sulfate assimilation in poplars (Populus
tremula x P. alba) overexpressing g-glutamylcysteine syn-
thetase in the cytosol. J Exp Bot 55: 837–845
Hatch MD and Osmond CB (1976) Compartmentation and
transport in C4 photosynthesis. In: Stocking CR and Heber
U (eds) Encyclopedia of Plant Physiology, New Series,
Vol 3, pp 144–184. Springer-Verlag, Berlin
Hell R and Bergmann L (1990) g-glutamylcysteine syn-
thetase in higher plants: catalytic properties and subcel-
lular localisation. Planta 180: 603–312
Herschbach C and Rennenberg H (2001) Significance of
phloem-translocated organic sulfur compounds for the
regulation of sulfur nutrition. Prog Bot 62: 177–192
Hirai MY, Fujiwara T, Awazuhara M, Kimura T, Noji M and
Saito K (2003) Global expression profiling of sulphur-
starved Arabidopsis by DNA macroarray reveals the role
of O-acetyl-L-serine as a general regulator of gene expres-
sion in response to sulphur nutrition. Plant J 33: 651–663
Hirai MY, Klein M, Fujikawa Y, Yano M, Goodenowe
DB, Yamazaki Y, Kanaya S, Nakamura Y, Kitayama M,
Suzuki H, Sakurai N, Shibata D, Tokuhisa J, Reichelt M,
Gershenzon J, Papenbrock J and Saito K (2005) Eluci-
dation of gene-to-gene and metabolite-to-gene networks
in Arabidopsis by integration of metabolomics and tran-
scriptomics. J Biol Chem 280: 25590–25595
Hirel B, Layzell DB, McCashin B, McNally SF and Canvin
DT (1983) Isoforms of glutamine synthetase in Panicum
species having C3, C4, and intermediate photosynthetic
pathways. Can J Bot 61: 2257–2259
Hirel B, Bertin P, Quillere I, Bourdoncle W, Attagnant C,
Dellay C, Gouy A, Cadiou S, Retailliau C, Falque M and
Gallais A (2001) Towards a better understanding of the
genetic and physiological basis for nitrogen use efficiency
in maize. Plant Physiol 125: 1258–1270
Hopkins L, Parmar S, Bouranis DL, Howarth JR and
Hawkesford MJ (2004) Coordinated expression of sulfate
uptake and components of the sulfate assimilatory path-
way in maize. Plant Biol 6: 408–414
Hopkins L, Parmar S, Błaszczyk A, Hesse H, Hoefgen R and
Hawkesford MJ (2005) O-acetylserine and the regulation of
expression of genes encoding components for sulfate uptake
and assimilation in potato. Plant Physiol 138: 433–440
Hothorn M, Wachter A, Gromes R, Stuwe T, Rausch T and
Scheffzek K (2006) Structural basis for the redox con-
trol of plant glutamate cysteine ligase. J Biol Chem 281:
27557–27565
Hylton CM, Rawsthorne S, Smith AM, Jones DA and Wool-
house HW (1988) Glycine decarboxylase is confined to
the bundle-sheath cells of leaves of C3-C4 intermediate
species. Planta 175: 452–459
Inokuchi R, Kuma KI, Miyata T and Okada M (2002)
Nitrogen-assimilating enzymes in land plants and algae:
7 Nitrogen and Sulfur in C4 Plants
phylogenic and physiological perspectives. Physiol Plant
116: 1–11
Jez JM, Cahoon RE and Chen S (2004) Arabidopsis thaliana
glutamate-cysteine ligase: functional properties, kinetic
mechanism, and regulation of activity. J Biol Chem 279:
33463–33470
Jost R, Altschmied L, Bloem E, Bogs J, Gershenzon J, Hähnel
U, Hänsch R, Hartmann T, Kopriva S, Kruse C,
Mendel RR, Papenbrock J, Reichelt M, Rennenberg H,
Schnug E, Schmidt A, Textor S, Tokuhisa J, Wachter A,
Wirtz M, Rausch T, and Hell R (2005) Expression profiling
of metabolic genes in response to methyl jasmonate reveals
regulation of genes of primary and secondary sulfur-related
pathways in Arabidopsis thaliana. Photosynth Res 36:
491–508
Kaiser WM and Huber SC (2001) Post-translational regu-
lation of nitrate reductase: mechanism, physiological
relevance and environmental triggers. J Exp Bot 52:
1981–1989
Ketchner SL and Sayre RT (1992) Characterization of the
expression of the photosystem II-oxygen evolving complex
in C4 species of Flaveria. Plant Physiol 98: 1154–1162
Kocsy G, Brunner M, Rüegsegger A, Stamp P and Brunold
C (1996) Glutathione synthesis in maize genotypes with
different sensitivities to chilling. Planta 198: 365–370
Kocsy G, Owttrim G, Brander K and Brunold C (1997) Effect
of chilling on the diurnal rhythm of enzymes involved in
protection against oxidative stress in a chilling-tolerant
and a chilling-sensitive maize genotype. Physiol Plant 99:
249–254
Kocsy G, Szalai G, Vagujfalvi A, Stehli L, Orosz G and
Galiba G (2000a) Genetic study of glutathione accumula-
tion during cold hardening in wheat. Planta 210: 295–301
Kocsy G, von Ballmoos P, Suter M, Ruegsegger A, Galli U,
Szalai G, Galiba G and Brunold C (2000b) Inhibition of
glutathione synthesis reduces chilling tolerance in maize.
Planta 211: 528–536
Kocsy G, Galiba G and Brunold C (2001) Role of glutath-
ione in adaptation and signalling during chilling and cold
acclimation in plants. Physiol Plant 113: 158–164
Kopriva S (2006) Regulation of sulfate assimilation in Ara-
bidopsis and beyond. Ann Bot 97: 479–495
Kopriva S and Koprivova A (2005) Sulfate assimilation and
glutathione synthesis in C4 plants. Photosynth Res 86:
363–372
Kopriva S and Rennenberg H (2004) Control of sulphate
assimilation and glutathione synthesis: interaction with N
and C metabolism. J Exp Bot 55: 1831–1842
Kopriva S, Chu C-C and Bauwe H (1996) Molecular
phylogeny of Flaveria as deduced from the analysis of
H-protein nucleotide sequences. Plant Cell Environ 19:
1028–1036
Kopriva S, Muheim R, Koprivova A, Trachsler N, Cata-
lano C, Suter M and Brunold C (1999) Light regulation
of assimilatory sulfate reduction in Arabidopsis thaliana.
Plant J 20: 37–44
125
Kopriva S, Jones S, Koprivova A, Suter M, von Ballmoos P,
Brander K, Flückiger J and Brunold C (2001) Influence of
chilling stress on the intercellular distribution of assimila-
tory sulfate reduction and thiols in Zea mays. Plant Biol
3: 24–31
Kopriva S, Suter M, von Ballmoos P, Hesse H, Krähenbühl
U, Rennenberg H and Brunold C (2002) Interaction of
sulfate assimilation with carbon and nitrogen metabolism
in Lemna minor. Plant Physiol 130: 1406–1413
Koprivova A, Suter M, Op den Camp R, Brunold C and
Kopriva S (2000) Regulation of sulfate assimilation by
nitrogen in Arabidopsis. Plant Physiol 122: 737–746
Koprivova A, Melzer M, von Ballmoos P, Mandel T, Brunold
C and Kopriva S (2001) Assimilatory sulfate reduction in
C3, C3-C4, and C4 species of Flaveria. Plant Physiol 127:
543–550
Kruse J, Hetzger I, Hänsch R, Mendel RR, Walch-Liu P,
Engels C, and Rennenberg H (2002) Elevated pCO(2 )
favours nitrate reduction in the roots of wild-type tobacco
(Nicotiana tabacum cv. Gat.) and significantly alters
N-metabolism in transformants lacking functional nitrate
reductase in the roots. J Exp Bot 53: 2351–2367
Ku MSB, Wu JR, Dai ZY, Scott RA, Chu C and Edwards
GE (1991) Photosynthetic and photorespiratory character-
istics of Flaveria species. Plant Physiol 96: 518–528
Lappartient AG and Touraine B (1996) Demand-driven con-
trol of root ATP sulphurylase activity and SO42- uptake
in intact canola. The role of phloem-translocated glutath-
ione. Plant Physiol 111: 147–157
Lejay L, Tillard P, Lepetit M, Domingo Olive F, Filleur S,
Daniel-Vedele F and Gojon A (1999) Molecular and func-
tional regulation of two NO3− uptake systems by N- and
C-status of Arabidopsis plants. Plant J 18: 509–519
Lejay L, Gansel X, Cerezo M, Tillard P, Muller C, Krapp
A, von Wiren N, Daniel-Vedele F and Gojon A (2003)
Regulation of root ion transporters by photosynthesis:
functional importance and relation with hexokinase. Plant
Cell 15: 2218–2232
Leustek T, Martin MN, Bick JA and Davies JP (2000)
Pathways and regulation of sulfur metabolism revealed
through molecular and genetic studies. Annu Rev Plant
Physiol Plant Mol Biol 51: 141–165
Lillo C, Meyer C, Lea US, Provan F and Oltedal S (2004)
Mechanism and importance of post-translational regula-
tion of nitrate reductase. J Exp Bot 55: 1275–1282
Linka M and Weber AP (2005) Shuffling ammonia between
mitochondria and plastids during photorespiration. Trends
Plant Sci 10: 461–465
Loque D and von Wiren N (2004) Regulatory levels for
the transport of ammonium in plant roots. J Exp Bot 55:
1293–1305
Lunn J, Droux M, Martin J and Douce R (1990) Localiza-
tion of ATP sulfurylase and O-acetylserine (thiol)lyase in
spinach leaves. Plant Physiol 94: 1345–1352
Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C,
Dubois F, Balliau T, Valot B, Davanture M, Terce-Laforgue
126
T, Quillere I, Coque M, Gallais A, Gonzalez-Moro MB,
Bethencourt L, Habash DZ, Lea PJ, Charcosset A, Perez
P, Murigneux A, Sakakibara H, Edwards KJ and Hirel B
(2006) Two cytosolic glutamine synthetase isoforms of
maize are specifically involved in the control of grain
production. Plant Cell 18: 3252–3274
Maruyama-Nakashita A, Nakamura Y, Watanabe-Takahashi A,
Inoue E, Yamaya T and Takahashi H (2005) Identification
of a novel cis-acting element conferring sulfur deficiency
response in Arabidopsis roots. Plant J 42: 305–314
Maruyama-Nakashita A, Nakamura Y, Tohge T, Saito K and
Takahashi H (2006) Arabidopsis SLIM1 is a central tran-
scriptional regulator of plant sulfur response and metabo-
lism. Plant Cell 18: 3235–3251
Matsumura T, Kimata-Ariga Y, Sakakibara H, Sugiyama
T, Murata H, Takao T, Shimonishi Y and Hase T (1999)
Complementary DNA cloning and characterization of
ferredoxin localized in bundle-sheath cells of maize
leaves. Plant Physiol 119: 481–488
May MJ, Vernoux T, Leaver C, van Montagu M and Inzé D
(1998a) Glutathione homeostasis in plants: implications
for environmental sensing and plant development. J Exp
Bot 49: 649–667
May MJ, Vernoux T, Sanchez-Fernandez R, Van Montagu
M and Inzé D (1998b) Evidence for posttranscriptional
activation of gamma-glutamylcysteine synthetase dur-
ing plant stress responses. Proc Natl Acad Sci USA 95:
12049–12054
McNally SF, Hirel B, Gadal P, Mann AF and Stewart GR
(1983) Glutamine synthetases of higher plants: evidence
for a specific isoform content related to their possible
physiological role and their compartmentation within the
leaf. Plant Physiol 72: 22–25
Mellor GE and Tregunna EB (1971) The localization of
nitrate-assimilating enzymes in leaves of plants with the
C4-pathway of photosynthesis. Can J Bot 49: 137–142
Meyer AJ and Fricker MD (2000) Direct measurement of
glutathione in epidermal cells of intact Arabidopsis roots
by two-photon laser scanning microscopy. J Microsc 198:
174–181
Meyer AJ, May MJ and Fricker M (2001) Quantitative
in vivo measurement of glutathione in Arabidopsis cells.
Plant J 27: 67–78
Miflin BJ (1972) The role of light in nitrite reduction: stud-
ies with leaf disks. Planta 105: 225–233
Miflin BJ and Habash DZ (2002) The role of glutamine syn-
thetase and glutamate dehydrogenase in nitrogen assimi-
lation and possibilities for improvement in the nitrogen
utilization of crops. J Exp Bot 53: 979–987
Miller AJ, Fan X, Orsel M, Smith SJ and Wells DM (2007)
Nitrate transport and signalling. J Exp Bot 58: 2297–2306
Mok DWS and Mok MC (2001) Cytokinin metabolism and
action. Annu Rev Plant Physiol Plant Mol Biol 52: 89–118
Monson RK and Moore BD (1989) On the significance of
C3-C4 intermediate photosynthesis to the evolution of C4
photosynthesis. Plant Cell Environ 12: 689–699
Stanislav Kopriva
Monson RK, Moore BD, Ku MSB and Edwards GE (1986)
Co-function of C3- and C4-photosynthetic pathways in C3,
C4 and C3-C4 intermediate Flaveria species. Planta 168:
493–502
Moore R and Black CC Jr (1979) Nitrogen assimilation
pathways in leaf mesophyll and bundle sheath cells of C4
photosynthesis plants formulated from comparative stud-
ies with Digitaria sanguinalis (L.) Scop. Plant Physiol 64:
309–313
Mullineaux PM and Rausch T (2005) Glutathione, photo-
synthesis and the redox regulation of stress-responsive
gene expression. Photosynth Res 86: 459–474
Neuenschwander U, Suter M and Brunold C (1991) Regula-
tion of sulfate assimilation by light and O-acetyl-L-serine
in Lemna minor L. Plant Physiol 97: 253–258
Noctor G and Foyer CH (1998) Ascorbate and glutathione:
keeping active oxygen under control. Annu Rev Plant
Physiol Plant Mol Biol 49: 249–279
Noctor G, Strohm M, Jouanin L, Kunert KJ, Foyer CH and
Rennenberg H (1996) Synthesis of glutathione in leaves
of transgenic poplar overexpressing g-glutamylcysteine
synthetase. Plant Physiol 112: 1071–1078
Noctor G, Arisi A-CM, Jouanin L, Kunert KJ, Rennen-
berg H and Foyer CH (1998a) Glutathione: biosynthesis,
metabolism and relationship to stress tolerance explored
in transformed plants. J Exp Bot 49: 623–647
Noctor G, Arisi A-CM, Jouanin L and Foyer CH (1998b)
Manipulation of glutathione and amino acid biosynthesis
in the chloroplast. Plant Physiol 118: 471–482
Noctor G, Arisi A-CM, Jouanin L and Foyer CH (1999)
Photorespiratory glycine enhances glutathione accumula-
tion in both the chloroplastic and cytosolic compartments.
J Exp Bot 50: 1157–1167
Nussbaum S, Schmutz K and Brunold C (1988) Regulation of
assimilatory sulfate reduction by cadmium in Zea mays L.
Plant Physiol 88: 1407–1410
Ohkama N, Takei K, Sakakibara H, Hayashi H, Yoneyama
T and Fujiwara T (2002) Regulation of sulfur-responsive
gene expression by exogenously applied cytokinins in
Arabidopsis thaliana. Plant Cell Physiol 43: 1493–1501
Oliveira IC and Coruzzi GM (1999) Carbon and amino acids
reciprocally modulate the expression of glutamine syn-
thetase in Arabidopsis. Plant Physiol 121: 301–310
Papen H, Gessler A, Zumbusch E and Rennenberg H (2002)
Chemolithoautotrophic nitrifiers in the phyllosphere of
a spruce ecosystem receiving high atmospheric nitrogen
input. Curr Microbiol 44: 56–60
Parisy V, Poinssot B, Owsianowski L, Buchala A, Glaze-
brook J, and Mauch F (2007) Identification of PAD2 as a
gamma-glutamylcysteine synthetase highlights the impor-
tance of glutathione in disease resistance of Arabidopsis.
Plant J 49: 159–172
Passera C and Ghisi R (1982) ATP sulphurylase and
O-acetylserine sulphydrylase in isolated mesophyll pro-
toplasts and bundle sheath strands of S-deprived maize
leaves. J Exp Bot 33: 432–438
7 Nitrogen and Sulfur in C4 Plants
Pastori GM, Mullineaux PM and Foyer CH (2000) Post-
transcriptional regulation prevents accumulation of
glutathione reductase protein and activity in the bundle
sheath cells of maize. Plant Physiol 122: 667–675
Persson J, Högberg P, Ekblad A, Högberg MN, Nordgren
A and Näsholm T (2003) Nitrogen acquisition from inor-
ganic and organic sources by boreal forest plants in the
field. Oecologia 137 :252–257
Prior A, Uhrig JF, Heins L, Wiesmann A, Lillig CH, Stoltze C,
Soll J and Schwenn JD (1999) Structural and kinetic prop-
erties of adenylyl sulfate reductase from Catharanthus
roseus cell cultures. Biochim Biophys Acta 1430: 25–38
Rathnam CKM and Das VSR (1974) Nitrate metabolism in
relation to the aspartate-type C-4 pathway of photosyn-
thesis in Eleusine coracana. Can J Bot 52: 2599–2605
Rathnam CKM and Edwards GE (1976) Distribution of
nitrate-assimilating enzymes between mesophyll proto-
plasts and bundle sheath cells in leaves of three groups of
C4 plants. Plant Physiol 57: 881–885
Rausch T and Wachter A (2005) Sulfur metabolism: a ver-
satile platform for launching defence operations.Trends
Plant Sci 10: 503–509
Rawsthorne S (1992) C3-C4 intermediate photosynthesis –
linking physiology to gene expression. Plant J 2: 267–274
Remans T, Nacry P, Pervent M, Filleur S, Diatloff E, Mounier
E, Tillard P, Forde BG and Gojon A (2006) The Arabidop-
sis NRT1.1 transporter participates in the signaling path-
way triggering root colonization of nitrate-rich patches.
Proc Natl Acad Sci USA 103: 19206–19211
Ritenour GL, Joy KW, Bunning J and Hageman RH. (1966)
Intracellular localization of nitrate reductase, nitrite
reductase, and glutamic acid dehydrogenase in green leaf
tissue. Plant Physiol 42: 233–237
Rotte C and Leustek T (2000) Differential subcellular
localization and expression of ATP sulfurylase and
5¢-adenylylsulfate reductase during ontogenesis of Ara-
bidopsis leaves indicates that cytosolic and plastid forms
of ATP sulfurylase may have specialised functions. Plant
Physiol 124: 715–724
Ruegsegger A and Brunold C (1992) Effect of cadmium on
g-glutamylcysteine synthesis in maize seedlings. Plant
Physiol 99: 428–433
Ruegsegger A and Brunold C (1993) Localization of [gamma]-
glutamylcysteine synthetase and glutathione synthetase
activity in maize seedlings. Plant Physiol 101: 561–566
Ruegsegger A, Schmutz D and Brunold C (1990) Regulation
of glutathione synthesis by cadmium in Pisum sativum L.
Plant Physiol 93: 1579–1584
Rufty TW Jr, MacKown CT and Volk RJ (1989) Effects of
altered carbohydrate availability on whole-plant assimi-
lation of 15NO3−. Plant Physiol 89: 457–463
Saito K (2004) Sulfur assimilatory metabolism. The long
and smelling road. Plant Physiol 136: 2443–2450
Sanchez-Fernandez R, Fricker M, Corben LB, White NS,
Sheard N, Leaver CJ, Van Montagu M, Inze D and May
MJ (1997) Cell proliferation and hair tip growth in the
127
Arabidopsis root are under mechanistically different
forms of redox control. Proc Natl Acad Sci USA 94:
2745–2750
Santi S, Locci G, Monte R, Pinton R, and Varanini Z
(2003) Induction of nitrate uptake in maize roots:
expression of a putative high-affinity nitrate transporter
and plasma membrane H+-ATPase isoforms. J Exp Bot
54: 1851–1864
Schmutz D and Brunold C (1984) Intercellular localization
of assimilatory sulfate reduction in leaves of Zea mays
and Triticum aestivum. Plant Physiol 74: 866–870
Schmutz D and Brunold C (1985) Localization of nitrite and
sulfite reductase in bundle sheath and mesophyll cells of
maize leaves. Physiol Plant 64: 523–528
Schaefer HJ, Haag-Kerwer A and Rausch T (1998) cDNA
cloning and expression analysis of genes encoding
GSH synthesis in roots of the heavy-metal accumulator
Brassica juncea L.: evidence for Cd-induction of a puta-
tive mitochondrial gamma-glutamylcysteine synthetase
isoform. Plant Mol Biol 37: 87–97
Sheen J (1999) C4 gene expression. Annu Rev Plant Physiol
Plant Mol Biol 50: 187–217
Stanford C, Larsen K, Barker DG and Cullimore JV (1993)
Differential expression within the glutamine synthetase
gene family of the model legume, Medicago truncatula.
Plant Physiol 103: 73–81
Stitt M, Muller C, Matt P, Gibon Y, Carillo P, Morcuende
R, Scheible WR and Krapp A (2002) Steps towards an
integrated view of nitrogen metabolism. J Exp Bot 53:
959–970
Strohm M, Jouanin L, Kunert KJ, Pruvost C, Polle A, Foyer
CH and Rennenberg H (1995) Regulation of glutathione
synthesis in leaves of transgenic poplar (Populus tremula
X P.alba) overexpressing glutathione synthetase. Plant J
7: 141–145
Suter M, von Ballmoos P, Kopriva S, Op den Camp R,
Schaller J, Kuhlemeier C, Schürmann P and Brunold C
(2000) Adenosine 5¢-phosphosulfate sulfotransferase
and adenosine 5¢-phosphosulfate reductase are identical
enzymes. J Biol Chem 275: 930–936
Tabe LM and Droux M (2002) Limits to sulfur accumulation
in transgenic lupin seeds expressing a foreign sulfur-rich
protein. Plant Physiol 128: 1137–1148
Taira M, Valtersson U, Burkhardt B and Ludwig RA. (2004)
Arabidopsis thaliana GLN2-encoded glutamine syn-
thetase is dual targeted to leaf mitochondria and chloro-
plasts. Plant Cell 16: 2048–2058
Tobin AK and Yamaya T (2001) Cellular compartmentation
of ammonium assimilation in rice and barley. J Exp Bot
52: 591–604
Vance CP (2001) Symbiotic nitrogen fixation and phospho-
rus acquisition. Plant nutrition in a world of declining
renewable resources. Plant Physiol 127: 390–397
Vauclare P, Kopriva S, Fell D, Suter M, Sticher L, von Ball-
moos P, Krähenbühl U, Op den Camp R and Brunold C
(2002) Flux control of sulphate assimilation in Arabidop-
 128
sis thaliana: Adenosine 5¢-phosphosulphate reductase is
more susceptible to negative control by thiols than ATP
sulphurylase. Plant J 31: 729–740
Vaughn KC and Campbell WH (1988) Immunogold locali-
zation of nitrate reductase in maize leaves. Plant Physiol
88: 1354–1357
Vernoux T, Wilson RC, Seeley KA, Reichheld JP, Muroy
S, Brown S, Maughan SC, Cobbett CS, Van Montagu M,
Inze D, May MJ and Sung ZR (2000) The ROOT MER-
ISTEMLESS1/CADMIUM SENSITIVE2 gene defines a
glutathione-dependent pathway involved in initiation and
maintenance of cell division during postembryonic root
development. Plant Cell 12: 97–109
von Arb C and Brunold C (1986) Enzymes of assimilatory
sulphate reduction in leaves of Pisum sativum: activity
changes during ontogeny and in vivo regulation by H2S
and cyst(e)ine. Physiol Plant 67: 81–86
von Wiren N, Gazzarrini S, Gojon A and Frommer WB
(2000) The molecular physiology of ammonium uptake
and retrieval. Curr Opin Plant Biol 3: 254–261
Wachter A, Wolf S, Steininger H, Bogs J and Rausch T
(2005) Differential targeting of GSH1 and GSH2 is
achieved by multiple transcription initiation: implications
for the compartmentation of glutathione biosynthesis in
the Brassicaceae. Plant J 41: 15–30
Wagner BM and Beck EH (1993) Cytokinins in the peren-
nial herb Urtica dioica L. as influenced by its nitrogen
status. Planta 190: 511–518
Walker MA and McKersie BD (1993) Role of the ascorbate-
glutathione antioxidant system in chilling resistance of
tomato. J Plant Physiol 141: 234–239
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Wang R, Tischner R, Gutierrez RA, Hoffman M, Xing X, Chen
M, Coruzzi G and Crawford NM (2004) Genomic analysis
of the nitrate response using a nitrate reductase-null mutant
of Arabidopsis. Plant Physiol 136: 2512–2522
Weber A and Flugge UI (2002) Interaction of cytosolic and
plastidic nitrogen metabolism in plants. J Exp Bot 53:
865–874
Westerman S, Stulen I, Suter M, Brunold C and De Kok JL
(2001) Atmospheric H2S as sulphur source for Brassica
oleracea: consequences for the activity of the enzymes of
the assimilatory sulphate reduction pathway. Plant Physiol
Biochem 39: 425–432
Wirtz M, Droux M and Hell R (2004) O-acetylserine (thiol)
lyase: an enigmatic enzyme of plant cysteine biosyn-
thesis revisited in Arabidopsis thaliana. J Exp Bot 55:
1785–1798
Xiang C and Oliver DJ (1998) Glutathione metabolic genes
coordinately respond to heavy metals and jasmonic acid
in Arabidopsis. Plant Cell 10: 1539–1550
Yonekura-Sakakibara K, Onda Y, Ashikari T, Tanaka Y,
Kusumi T and Hase T (2000) Analysis of reductant
supply systems for ferredoxin-dependent sulfite reductase
in photosynthetic and nonphotosynthetic organs of maize.
Plant Physiol 122: 887–894
Zechmann B, Zellnig G and Muller M (2005) Changes in the
subcellular distribution of glutathione during virus infec-
tion in Cucurbita pepo (L.). Plant Biol 7: 49–57
Zimmermann P, Hirsch-Hoffmann M, Hennig L and
Gruissem W (2004) GENEVESTIGATOR. Arabidopsis
microarray database and analysis toolbox. Plant Physiol
136: 2621–2632