Journal of Experimental Botany, Vol. 57, No. 8, pp.

16031620, 2006
Oxygen Metabolism, ROS and Redox Signalling in Plants Special Issue
doi:10.1093/jxb/erj202 Advance Access publication 12 May, 2006

NAD(P) synthesis and pyridine nucleotide cycling in plants

and their potential importance in stress conditions*

Graham Noctor , Guillaume Queval and Bertrand Gakie`re
Institut de Biotechnologie des Plantes, Universite de Paris XI, F-91405 Orsay, France

Received 1 March 2006; Accepted 17 March 2006

Abstract
Pyridine nucleotides are key redox carriers in the soluble
phase of all living cells, and both NAD and NADP play
crucial roles in pro-oxidant and antioxidant metabolism.
Recent data also suggest a number of non-redox
mechanisms by which these nucleotides could influence
cell function. In cases where these mechanisms involve
NAD(P) consumption, resynthesis must occur to maintain
nucleotide pools. Important in-formation on the pathways
involved in NAD synthesis in plants is beginning to
appear, but many outstanding questions remain. This
work provides an overview of the current state of
knowledge on NAD synthesis path-ways in plants and
other organisms, analyses plant sequences for the first
two enzymes of the de novo synthesis of NAD, proposes
a preliminary model for the intracellular distribution of
NAD synthesis, pres-ents plant homologues of recently
identified yeast mitochondrial NAD transporters, and
discusses factors likely to be important in the regulation of
NAD synthe-sis and contents in plants, with particular
reference to stress conditions.
Key words: L-Aspartate oxidase, compartmentation, NAD
trans-porter, NAD signalling, nicotinamide, nicotinate,
quinolinate synthase.
Introduction
NAD and its derivative NADP are ubiquitous in living cells
(Penfound and Foster, 1996; Kurnasov et al., 2003). They
mediate hundreds of redox reactions, and thus impact on
virtually every metabolic pathway in the cell. Pyridine
nucleotides are also key players in signalling through
reactive oxygen species (ROS) since they are crucial in the
regulation of both ROS-producing and ROS-consuming
systems in plants (Mller, 2001; Apel and Hirt, 2004; Mittler
et al., 2004; Foyer and Noctor, 2005). Chloroplast ROS
+
production is influenced by NADP :NADPH ratios,
mitochondrial NAD(P) status is important in determining
ROS formation by the respiratory electron transport chain,
and NAD(P)H oxidases are key players in the generation of
ROS at the plasmalemma. Similarly, ROS processing partly
depends on ascorbate and glutathione, and redox flux
through these pools is maintained by NAD(P)H. Pyridine
nucleotides are involved in many other defence and
signalling reactions, for example, production of nitric oxide
and metabolism of reactive lipid derivatives (Crawford and
Guo, 2005; Mano et al., 2005). Thus, as well as their roles as
cofactors in numerous energy-producing and bio-synthetic
reactions, NAD and NADP are crucial in redox signalling
linked to stress and development.
Recent studies show that NAD and NADP play import-ant
roles in cell signalling in animals, plants, and fungi (Hunt et
al., 2004; Ziegler, 2005). NAD and NADP are the respective
substrates for the production of the calcium agonists cyclic
ADP-ribose (cADP-R; Sanchez et al., 2004) and nicotinate
adenine dinucleotide phosphate (NaADP; Navazio et al.,
2000; Lee, 2005). Transfer of ADP-ribose

* In this article, NAD(P) is used to denote total pools of reduced and oxidized forms or in cases where the distinction between the two forms is
unnecessary (e.g. NAD synthesis). NAD(P) + is used to make specific reference to the oxidized forms.
y
To whom correspondence should be addressed. E-mail: noctor@ibp.u-psud.fr
Abbreviations: (c)ADP-R, (cyclic)ADP-ribose; CMS, cytoplasmic male sterile; DHAP, dihydroxyacetone phosphate; MNam, N-methyl nicotinamide; NaAD,
nicotinate adenine dinucleotide; NaADP, nicotinate adenine dinucleotide phosphate; NADase, NAD glycohydrolase; NaMN(AT), nicotinate
mononucleotide (adenylyl transferase); NaPRT, nicotinate phosphoribosyltransferase; NMDA, N-methyl- D-aspartate; NMN(AT), nicotinamide
mononucleotide (adenylyl transferase); NRK, nicotinamide riboside kinase; PARP, poly(ADP-ribose) polymerase; QPRT, quinolinate
phosphoribosyltransferase; ROS, reactive oxygen species; SIRTUIN, homologue of the yeast Silent Information Regulator 2 protein.

The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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1604 Noctor et al.
units from NAD to target proteins is an important post-
translational modification, catalysed by poly(ADP-ribose)
polymerases (PARPs) and mono(ADP-ribosyl)transferases
(Ying et al., 2003; Graziani et al., 2005). The yeast Silent
Information Regulator 2 (Sir2) protein has been shown to be
an NAD-dependent deacetylase (Imai et al., 2000; Lin et al.,
2000; Berger et al., 2004; Ziegler, 2005). Although these
components and phenomena are better characterized in other
systems than in plants, many of the products and the
enzymes that produce them are also found in plants (Hunt et
al., 2004). Since some of these NAD(P)-consuming reactions
are important in stress conditions and/or are stress inducible,
NAD concentration could be influential in plant responses to
environmental challenge. Exposure of plant cells to
oxidative stress produces decreases in overall NAD contents,
linked to PARP activity (Amor et al., 1998; De Block et al.,
2005). Recent studies of mutants provide evidence that
changes in NAD status can alter photosynthesis and plant
stress responses (Dutilleul et al., 2003a, b; Hayashi et al.,
2005), and suggest that NAD content could be a powerful
modulator of metabolic integration (Dutilleul et al., 2005).
Much remains to be discovered about the factors controlling
pyridine nucleo-tide synthesis and recycling in plants.
NAD recycling and associated reactions
The most studied route to NAD in mammals is via
nicotinamide and nicotinate. Together, these compounds are
called niacins and were identified by their ability to prevent
pellagra in dogs fed a diet poor in tryptophan, which is a
precursor in the de novo synthesis of the pyridine ring in
animals (see below). It was subsequently shown that niacins
can be converted to NAD via nicotinate phosphoribosyl-
transferase (NaPRT), nicotinate mononucleotide adenylyl-
transferase (NaMNAT), and NAD synthetase (Preiss and
Handler, 1958). Recently, the structure of a yeast NaPRT
has been determined at a resolution of 1.75 A (Chappie et
al., 2005). In animals, which lack nicotinamide de-
amidase activity (Chappie et al., 2005), NAD can also be
produced via nicotinamide phosphoribosyltransferase
(NPRT) activity, followed by adenylylation of NMN. This
allows incorporation of nicotinamide into NAD in two
steps rather than the four-step pathway via nicotinate (Fig.
1, orange background).
Labelling studies in plants suggest that nicotinamide
released from NAD is first deamidated by nicotinamidases to
nicotinate (Ryrie and Scott, 1969; Ashihara et al., 2005;
Zheng et al., 2005) which is then converted to NaMN by
NaPRT (Fig. 1). This view is supported by the presence in
Arabidopsis and other plants of genes predicted to encode
NaPRT, NaMNAT, and NAD synthetase (Hunt et al., 2004;
Katoh and Hashimoto, 2004). Consistent with this,
homologues of mammalian NPRT gene sequences appear to
be absent from plants (Katoh and Hashimoto, 2004) and
NPRT activity has not been detected in extracts of
tobacco, mungbean, or white spruce cells (Wagner et al.,
1986; Ashihara et al., 2005; Zheng et al., 2005).
14
Results of a detailed study of the fate of C-labelled
nicotinamide in coffee and cocao (Zheng et al., 2004) also
suggest that nicotinate is the main precursor of pyridine
nucleotides, although NMN was also labelled in signifi-cant
amounts, probably as a breakdown product of NAD through
NAD pyrophosphatase activity (Fig. 1). In view of these
results, together with data gathered from labelling studies in
white spruce and mungbean (Ashihara et al., 2005; Zheng et
al., 2005), three possible routes were suggested from NAD
to nicotinamide. The first involves production of
nicotinamide in a single step, with ADP-R (or derivatives) as
the other product. This route would result from the activities
of enzymes such as NAD glyco-hydrolase, PARP,
SIRTUINS, and ADP-ribosyl cyclase (Fig. 1). A second
pathway would proceed via NMN and involve two steps,
catalysed by NAD pyrophosphatase and NMN nucleosidase
(Zheng et al., 2004). In the third pathway, NMN is indirectly
converted to nicotinamide via nicotinamide riboside, and
overall the three steps involve NAD pyrophosphatase, 5
nucleotidase, and nicotinamide nucleoside phosphorylase or
nucleosidase (Ashihara et al., 2005; Zheng et al., 2005).
According to this scenario, full turnover of the pyridine
cycling pathway (from NAD to NAD) could involve five,
six, or seven steps. The final four steps would be common to
all these recycling pathways and involve formation of
nicotinate from nico-tinamide, conversion of nicotinate to
NaMN, adenylylation of NaMN to NaAD (desamidoNAD),
and amidation of NaAD to produce NAD (Fig. 1).
Given the presence of NMN in plant tissues, further work
is required to confirm the substrate specificities of plant
N(a)MNAT activities, and to establish whether these en-
zymes are specific to NaMN. In mammals, three adenylyl-
transferases have been characterized and all are able to use
NaMN and NMN (Magni et al., 2004). This probably
reflects the dual role of mammalian N(a)MNAT in both the
de novo pathway (production of NaAD from NaMN) and the
recycling pathway (production of NAD from NMN; Fig. 1,
orange background, top). In bacteria, the adenylyltransferase
shows a clear preference for NaMN, a compound which in
these organisms is common to both de novo synthesis and
the recycling pathway from nicotin-amide by the successive
action of the pncA and pncB gene products (Fig. 1). The
final step of NAD synthesis is cat-alysed by NAD
synthetases, probably using glutamine, although in some
organisms isoforms are able to use am-monia under
physiological conditions (Magni et al., 2004).
In addition to synthesis of pyridine mononucleotides
(NMN and NaMN) by phosphoribosyltransferase activities,
+
a third path to NAD has been described in species of
bacteria, yeast, and mammals (Bieganowski and Brenner,
2004). This route involves synthesis of NMN by

phosphorylation of nicotinamide riboside (Fig. 1). An
elegant vitamin screen using yeast mutants indicated the
presence of nicotinamide riboside in milk (Bieganowski and
Brenner, 2004). As discussed above, nicotinamide riboside
might be formed in mungbean from NMN (Zheng et al.,
2005). A BLAST search of several plant genome databases
using bacterial, yeast, and mammalian sequences for
nicotinamide riboside kinase revealed no fully hom-ologous
sequences, although homologous domains can be identified
among the uridine kinase family.
In addition to nicotinamide riboside, plants contain
nicotinate riboside. Indeed, labelling studies in coffee
have provided some evidence that this compound is an
intermediate in pyridine cycling and that NaMN can be
formed from nicotinate through two paths (Zheng et al.,
2004). As well as direct phosphoribose transfer catalysed
by NaPRT, a second path is possible through successive
ribosylation and phosphorylation (Fig. 1, orange back-
ground, bottom). These reactions are reversible and could
also represent a route from NaMN to nicotinate (Ashihara
et al., 2005). If they operate in the direction of NaMN
synthesis, it would add yet another step to the recycling
pathway in plants. In this case, recycling of the pyridine
ring from NAD to NAD could potentially consist of eight
steps rather than the five-step pathway described in
bacteria (Fig. 1, orange background, cf. shorter route
traced by the solid arrows with the longer path including
dotted arrows via the two ribosides).
Methylation of nicotinamide and nicotinate produces N-
methyl nicotinamide (MNam) and N-methyl nicotinate
(trigonelline). In mammals, MNam is the initial product of a
degradation pathway in which pyridone products are
subsequently formed by MNam oxidation (Fig. 1, grey
background). Urinary excretion of both MNam and pyri-
done products is stimulated when the diet is rich in
nicotinamide or nicotinate (Creeke and Seal, 2005, and
references therein). MNam has been detected in plants
(Waller et al., 1966) while trigonelline is known to be
formed in many types of plants, notably legumes, and may
have hormonal-type functions and be important as a store of
nicotinate (Minorsky, 2002; Zheng et al., 2004, 2005). In
addition to MNam and trigonelline, other plant compounds
could act as conjugated stores of nicotin-amide and
nicotinate, including N-glucosylated forms and nicotinate
riboside (Berglund et al., 1996; Zheng et al., 2004). In coffee
14
and cocoa, most of the label from [ C]nicotinamide was
incorporated into trigonelline, with only minor labelling of
nicotinate riboside and nicotinate glucoside (Zheng et al.,
2004). In contrast, in tea, the major labelled compound was
nicotinate glucoside, with no detectable label found in
trigonelline. These observations show that the type of
nicotinate conjugate formed is species dependent (Zheng et
al., 2004).
As well as trigonelline, other pyridine-based alkaloids
include ricinine, nicotine, and anabasine (Fig. 1, blue
NAD(P) synthesis and pyridine nucleotide cycling 1605
background). Production of these compounds is enhanced
by stress, particularly wounding, and they are effective
feeding deterrents. Much of the information that has been
gathered on the regulation of NAD-linked metabolism in
plants has come from investigations of pyridine alkaloid
synthesis rather than NAD(P) synthesis per se (Waller et
al., 1966; Frost et al., 1967; Mann and Byerrum, 1974a, b;
Wagner et al., 1986; Sinclair et al., 2000; Cane et al.,
2004; Zheng et al., 2004, 2005). Labelling studies have
shown that nicotinate, nicotinamide, quinolinate, NaAD,
and NAD are all effective sources of the pyridine ring for
nicotine and ricinine synthesis (Waller et al., 1966; Frost
et al., 1967). These data emphasize the close relationship
between NAD metabolism and alkaloid production in
these species. The observation that quinolinate and NAD
are as effective as nicotinate in supporting production of
pyridine alkaloids (Waller et al., 1966; Frost et al., 1967;
Zheng et al., 2005) suggests that, at least in those species
examined, the pyridine recycling pathway is capable of
relatively rapid turnover under many conditions.
NADP(H) synthesis and utilization
NADP is produced from NAD by NAD(H) kinase. At least
one NAD kinase in tobacco is a calmodulin-binding protein
(Harding et al., 1997) and activity in wild tomato may be
subject to regulation by thiol/disulphide exchange
(Delumeau et al., 2000). NAD kinase activity could be
located in the chloroplast, cytosol, and mitochondria (Gallais
et al., 2000a; Turner et al., 2004; Chai et al., 2005). Two
+
NAD -specific Arabidopsis NAD kinases, named NADK1
and NADK2, have been cloned and characterized
biochemically (Turner et al., 2004, 2005). Compared with
NADK1, NADK2 has a large N-terminal extension (Turner
et al., 2004), probably involved in re-gulation by calmodulin
(Harding et al., 1997). Total ex-tractable NAD kinase was
shown to be highly dependent on calmodulin in Arabidopsis
roots and shoots throughout development (Turner et al.,
2004). In contrast to NADK1 and NADK2, a third
characterized Arabidopsis isoform, NADK3, shows a strong
preference for NADH over NAD (60-fold lower Km for
NADH; Turner et al., 2005). The yeast homologue of
Arabidopsis NADK3 is a mitochondrial enzyme, whereas
the plant enzyme appears to be located in the cytosol (Turner
et al., 2005). In plants, mitochond-rial NADP could be at
least partly imported from the cytosol (Bykova and Mller,
2001). If NADK3 is a cyto-solic enzyme, some in vivo
+
activity with NAD cannot be completely discounted in view
+
of the high total NAD / NADH ratio in this compartment
(1030: Igamberdiev and Gardestro m, 2003). Indeed, the
+
physiologically relevant free NAD /NADH ratio
(nucleotides not bound to protein at any given moment) may
be as high as 6001000 in the cytosol of both plant and
mammalian cells (Heineke et al., 1991; Zhang et al., 2002).
Other authors have reported that
1606 Noctor et al.

Fig. 1. Principal reactions in NAD synthesis and metabolism. The scheme incorporates elements from current information on both plants and other organisms.
The solid arrows show the de novo synthesis pathway and the five-step NAD cycling pathway established in bacteria. In addition, a three-step recycling
pathway through NMN operates in animals, NAD can be synthesized from nicotinamide riboside in several organisms (Bieganowski and Brenner, 2004), and
it has been proposed that the cycling route in some plants could involve six, seven, or even eight steps (Zheng et al., 2004, 2005). Reactions necessary for
these pathways are shown as dotted arrows on the orange background. Some important pyridine alkaloids are shown against the blue background. Dashed
arrows show other routes to quinolinate apparently not present in plants. Bacterial nad [NAD(P) synthesis] and pnc (pyridine nucleotide cycle) homologue
names are indicated in parentheses for some of the enzymes. Abbreviations: AdoHcys, S-adenosylhomocysteine; AdoMet,
 NAD(P) synthesis and pyridine nucleotide cycling 1607

Arabidopsis NADK2 appears to localize to the chloro-plast
and that an insertional mutant for the enzyme shows stunted
growth and stress hypersensitivity, associated with a minor
+
decrease in total tissue NADPH (Chai et al., 2005). NADP
+ +
phosphatase activity, which converts NADP to NAD , has
also been reported in plants (Gallais et al., 2000b), and the
mitochondrial NAD(H) and NADP(H) pools are redox-
linked via transhydrogenases (Bykova et al., 1999).
As well as its redox cofactor function, NADP is the
precursor for the calcium channel activator NaADP (Lee,
2005). NaADP has been shown to be active in plants
(Navazio et al., 2000) and can be synthesized through base
exchange of the nicotinamide moiety of NADP with free
nicotinate, a reaction catalysed by ADP-ribosyl cyclases
under certain conditions (Fig. 1, green background, left).
NaADP concentrations could also be regulated by specific
calcium-dependent phosphatases, which convert it to NaAD
(Berridge et al., 2002), or nucleotide pyrophospha-tases,
which produce NaMN and AMP-29-phosphate (Evans et al.,
2005; Fig. 1). Glycohydrolases can function to degrade
NADP to nicotinamide and ADP-R 29-phosphate or cADP-
R 29-phosphate. The best-characterized ADP-ribosyl cyclase
in mammals is the membrane-bound glycoprotein CD38
(Evans et al., 2005). This protein can convert extracellular or
intracellular NAD to cADP-R, hydrolyse cADP-R to ADP-
R, and catalyse synthesis of NaADP via the base exchange
reaction with nicotinate (Fig. 1, green and grey
backgrounds). Although no genes have been identified on
the basis of sequence homology, low activities of both ADP-
ribosyl cyclase and cADP-R hydrolase have been detected in
Arabidopsis, and cADP-R has been shown to accumulate
transiently in response to abscisic acid (Sanchez et al.,
2004). In human platelets, it has also been reported that
CD38 can cleave NADP to ADP-R 29-phosphate (Torti et
al., 1999). Like the reactions catalysed by PARP and
SIRTUINS, many of these NAD and NADP cleavage
reactions produce nicotinamide (Fig. 1, green background).
De novo synthesis of nicotinate mononucleotide
(NaMN)
As well as producing NaMN from nicotinate obtained from
the environment or formed during the recycling pathway,
almost all organisms appear to be able to produce this NAD
precursor via synthesis of the pyridine ring in the form of
quinolinate (pyridine 2,3-dicarboxylate; Fig. 1, purple
background). NaMN is then produced by quinolinate
phosphoribosyltransferase (QPRT; EC 2.4.2.19), encoded by
nadC in Escherichia coli (Fig. 1). Characterization of the
enzyme purified from castor bean endosperm showed that
Km values for quinolinate and 5-phosphoribosyl-1-
pyrophosphate were 12 and 45 lM, respectively (Mann and
Byerrum, 1974a). Tobacco QPRT cDNA sequences
suggested that the protein is probably located in the
mitochondria, although a shorter transcript might produce a
cytosol-directed protein (Sinclair et al., 2000). Computer
analysis (http://urgi.infobiogen.fr/predotar) of sequences
encoded by the single QPRT gene in Arabidopsis (Fig. 1)
predicts two transcripts, the longer of which probably
encodes a mitochondrial import sequence. Quinolinate-
dependent synthesis of NaMN therefore probably occurs in
both cytosolic and mitochondrial compartments.
In animals and fungi, quinolinate is formed through a
multistep pathway from tryptophan. Recently, gene
sequences for several enzymes of this pathway have also
been found in certain pathogenic bacteria (Kurnasov et al.,
2003), and some anaerobic species have been reported to
make quinolinate by acetylation of N-formyl- L-aspartate
(Scott et al., 1969; Griffith et al., 1975). However, by far the
best characterized prokaryotic pathway for quinolinate
synthesis is from aspartate and triose phosphate (Griffith et
al., 1975; Flachmann et al., 1988; Kurnasov et al., 2003).
This pathway involves two enzymatic steps, catalysed by
quinolinate synthase, a term which has been used to refer to
the two enzymes functioning together or specifically to
designate the second enzyme. The first enzyme, L-aspartate
oxidase (or quinolinate synthase B), is encoded by nadB,
while the second, quinolinate synthase (or quinolinate
synthase A), is encoded by nadA (Fig. 1). Close asso-
ciation of the two enzymes might enable the intermediate,
iminoaspartate, an unstable compound that spontaneously
hydrolyses to oxaloacetate, to be channelled from the first
enzyme to the second. In E. coli, the nadB and nadA genes
are arranged in a single operon together with the nadC gene
encoding QPRT (Griffith et al., 1975).
Quinolinate synthesis in plants
In mammals, in addition to the well-known neuroactivity
of nicotine, the endogenously produced NAD precursor

S-adenosylmethionine; ADP-R cyc, ADP-ribosyl cyclase; (c)ADP-R, (cyclic)ADP-ribose; 29-P-(c)ADP-R, (cyclic)ADP-ribose-29-phosphate; 29-P-AMP,
1
AMP-29-phosphate; DHAP, dihydroxyacetone phosphate; MNam, N -methylnicotinamide; MRT, mono(ADP-ribose)transferase; NaAD, nicotinate adenine
dinucleotide; NaADP, nicotinate adenine dinucleotide phosphate; NADase, NAD glycohydrolase (NAD nucleosidase); NAD ppase, NAD pyrophosphatase;
+ +
NADP pase, NADP phosphatase; NAD-S, NAD synthetase; NaMN(AT), nicotinate mononucleotide (adenylyltransferase); NaPRT, nicotinate
phosphoribosyltransferase; NaRK, nicotinate riboside kinase; 59Nase, 59-nucleotidase; NMN(AT), nicotinamide mononucleotide (adenylyltransferase);
NMN nase, NMN nucleosidase; (N)NP, (nicotinamide) nucleoside phosphorylase; N ppase, nucleotide pyrophosphatase; NRK, nicotinamide riboside kinase;
PARP, poly(ADP-ribose)polymerase; 29 Pase, 29 phosphatase; Pi, phosphate; Ppi, pyrophosphate; Prot, protein; PRPP, 59-phosphoribosyl-19-pyrophosphate;
2-PYR, N-methyl-2-pyridone-5-carboxamide; 4-PYR, N-methyl-4-pyridone-3-carboxamide; QPRT, quinolinate phosphoribosyltransferase; R1P, ribose 1-
phosphate; SIRTUINS, homologues of the yeast Silent Information Regulator 2 (Sir2) protein. Not all possible interconversions are shown. Double-headed
arrows indicate some of the reactions that are theoretically reversible.
1608 Noctor et al.
quinolinate acts as an agonist at N-methyl- D-aspartate
(NMDA) receptors, which are classed among the glutamate
receptor family (Stone and Addae, 2002). Quinolinate
accumulation in the brain has been implicated in several
neurodegenerative disorders (Fukuoka et al., 2002). The
Arabidopsis genome contains numerous loci that encode
subunits of glutamate receptors. Although much remains to
be discovered concerning the functions of these proteins,
plant NMDA receptor-like proteins share certain character-
istics of their animal counterparts (Dubos et al., 2003). It is
an intriguing possibility that, in addition to NAD(P)-linked
signalling functions associated with redox control,
production of NaADP and cADP-ribose, and PARP and
SIRTUIN activities, other NAD(P) precursors or metab-
olites such as quinolinate might act as information trans-
mitters in plants.
To date, there have been very few studies of the enzymes
that produce quinolinate in plants, and it has often been
stated or implied that there is a strict prokaryotic/eukaryotic
division between quinolinate synthesis pathways (Garava-
glia et al., 2003; Bieganowski and Brenner, 2004) and thus
that plants use the tryptophan pathway (Hunt et al., 2004).
Indeed, the tryptophan pathway has been considered an
important route in several plants based on some labelling
studies (reviewed by Arditti and Tarr, 1979). However,
several investigations reported a lack of evidence for
quinolinate synthesis from tryptophan, for example, in maize
and tobacco (Henderson et al., 1958), and other authors have
thus favoured the aspartate pathway while noting that no
enzymes necessary to produce quinolinate had been found in
plants (Mann and Byerrum, 1974b). Subsequently, it was
reported that L-aspartate oxidase activity could be detected in
cotton callus cell extracts (Hosokawa et al., 1983) and, more
recently, analyses of gene sequences have reported the
presence in plant species of the bacterial pathway of
quinolinate synthesis from aspartate and triose phosphate
(Kurnasov et al., 2003; Katoh and Hashimoto, 2004).
Indeed, the Arabidopsis genome contains, as well as a single
gene predicted to encode QPRT, two sequences that show
significant hom-ology to the two bacterial enzymes involved
in this path-way (Fig. 1). In contrast, the Arabidopsis
genome does not appear to contain gene sequences that show
significant homology to tryptophan pathway enzymes. Other
authors have noted the presence of genes encoding enzymes
of both the aspartate and the tryptophan pathway in rice
(Katoh and Hashimoto, 2004), but sequences associated with
the latter pathway have been eliminated by subsequent
annotation updates of the genome. Likewise, no sequences
of signifi-cant homology are found in poplar, Medicago, or
Phys-comitrella databases. Moreover, a preliminary report
has described the cloning of Arabidopsis homologues for
nadA and nadB, their ability to rescue E. coli mutants
deficient in the respective enzymes, and the presence in
plastids of the encoded proteins (Katoh et al., 2005). These
data, together
with the apparent absence of genes encoding enzymes
involved in other possible routes to quinolinate, suggest that
plants obtain this key precursor via the aspartate path-way
used by many bacteria (Fig. 1, purple background).
An nadB homologue in plants
L-Aspartate oxidase (EC 1.4.3.16) catalyses the FAD-
dependent oxidation of L-aspartate to iminoaspartate (Fig. 1).
FAD is regenerated by either molecular oxygen or fumarate.
A single sequence is annotated L-aspartate oxidase in
Arabidopsis, and homologues are found in several other
plant species. Selected plant and bacterial protein sequences
were used to construct a phylogenetic tree that reveals a very
close relationship between proteins from angiosperm
species, with the moss sequence being classed slightly apart
(Fig. 2A). By contrast, the algal protein sequence is
somewhat intermediate between the bacterial and plant
proteins (Fig. 2A). Bacterial sequences cluster less tightly
than do the plant sequences, probably because the bacteria
for which sequences were analysed are found in very distinct
taxonomic groups.
A sequence alignment was performed to identify con-
served amino acid residues in L-aspartate oxidases from
different species (Fig. 2B). The proteins all contain two
large domains, one aspartate oxidase domain in the N-
terminal two-thirds of the protein and a succinate de-
hydrogenase domain in the C-terminal part (Mattevi et al.,
1999). Earlier studies of the cotton enzyme reported the
presence of a permanent cofactor with an apparent mol-
ecular mass of 1050 (Hosokawa et al., 1983). This co-
factor could be FAD, despite the latters smaller
molecular mass, as this nucleotide has been shown to be
essential to the bacterial enzyme. Recent crystallization of
the E. coli enzyme allied with site-directed mutagenesis
allowed identification of amino acid residues involved in
the FAD- and succinate-binding sites (Mattevi et al.,
1999; Bossi et al., 2001; Tedeschi et al., 2001). It is
noteworthy that these amino acids are strictly conserved
between plant and bacterial species (Fig. 2B), consistent
with the key role of the residues in the catalytic properties
of the en-zyme. Though not shown in Fig. 2B, an analysis
of plant sequences suggests the presence of a chloroplast
transit peptide in L-aspartate oxidase from Arabidopsis,
rice, poplar, and Medicago.
Certain organisms can oxidize aspartate to iminoaspar-
tate using enzymes other than L-aspartate oxidase. In
Thermogata maritima, the nadB gene encodes an NAD-
dependent aspartate dehydrogenase (EC 1.4.1.), an en-
zyme with a very different primary protein sequence from
L-aspartate oxidase (Yang et al., 2003). In mammals, a D-
aspartate oxidase (EC 1.4.3.1) synthesizes iminoaspartate,
and in vitro this enzyme can replace L-aspartate oxidase in
the quinolinate synthase complex (Nasu et al., 1982a). The
physiological role of this enzyme is not in NAD

synthesis but in regulating the concentration of D-
aspartate, a compound present in the brain and some
secretory organs, by converting it to iminoaspartate which
then decomposes to oxaloacetate (Wang et al., 2000;
Wolosker et al., 2000).
An nadA homologue in plants
Iminoaspartate produced by L-aspartate oxidase is com-bined
with dihydroxyacetone phosphate to produce quin-olinate, a
reaction catalysed by quinolinate synthase (also called
quinolinate synthase A). Sequences homologous to the
bacterial proteins were found in the Arabidopsis, rice, poplar,
Medicago, and Physcomitrella databases, but for
Chlamydomonas only a partial sequence is available. For the
purposes of sequence alignment, the analysis was restricted
to this part of the protein. Construction of a phylogenetic
tree for the protein sequences generates a clear distinction
between plants and bacteria (Fig. 3A). The five plant
sequences are closely related to each other and also to the
Chlamydomonas sequence. Bacterial sequences form a
cluster in which E. coli and Synechocystis are most closely
related. Plant and bacterial sequences appear more distinct
from each other than for L-aspartate oxidase.
Alignment of plant sequences with the partial Chlamy-
domonas sequence indicates the presence of several
highly conserved amino acid residues (Fig. 3B). The
three-dimensional structure of the crystallized quinolinate
syn-thase from Pyrococcus horikoshii shows a triangular
architecture in which conserved amino acids determine
three structurally homologous domains, termed three-
fold repeat of three-layer sandwich (Sakuraba et al.,
2005). Comparison of complete bacterial, rice, and
Arabidopsis sequences with the P. horikoshii sequence
suggests that all quinolinate synthases share this unique
triangular architecture, although the plant sequences
contain an additional C-terminal extension.
Recent studies demonstrate that E. coli quinolinate
synthase harbours a [4Fe4S] cluster (Cicchillo et al.,
2005; Ollagnier de Choudens et al., 2005). The sensitivity
of this cofactor to oxidation probably explains why quino-
linate synthase is the site of oxygen poisoning of pyridine
nucleotide synthesis in anaerobic bacteria (Gardner and
Fridovich, 1991; Draczynska-Lusiak and Brown, 1992;
Ollagnier de Choudens et al., 2005). The characteristic
binding motif for [4Fe4S] clusters is Cys-w-x-Cys-y-z-
Cys, and this is found in the C-terminal region of most
quinolinate synthases from bacteria (Ollagnier de
Choudens et al., 2005), including Synechocystis. No
homologous sequence is found in the plant quinolinate
synthase. The catalytic mechanism and cofactors for the
plant enzyme remain to be identified.
Although less clear for other available plant quinolinate
synthase sequences, the Arabidopsis and poplar enzymes
NAD(P) synthesis and pyridine nucleotide cycling 1609
are predicted to contain an N-terminal plastid targeting
peptide. Together with the chloroplastic localization of L-
aspartate oxidase, this suggests that quinolinate synthe-sis
probably occurs in this organelle. As iminoaspartate is
unstable, L-aspartate oxidase and quinolinate synthase could
occur in close association, although no conclusive evidence
has yet been found in bacteria for the formation of a
complex (Sakuraba et al., 2005).
Compartmentation and transport in
NAD synthesis and metabolism
Total NAD(H) concentrations in leaf cell mitochondria and
cytosol are ;2 and 0.6 mM, respectively, while total
NADP(H) pools are of the order of 0.3 mM in both
compartments (Igamberdiev and Gardestro m, 2003).
NAD(H) and NADP(H) pools of isolated chloroplasts have
both been calculated to be ;0.4 mM (Takahama et al., 1981).
Because of their key roles in metabolism in these
compartments and in others such as the peroxisomes, NAD
and NADP show very rapid redox turnover during processes
such as photosynthesis, photorespiration, and respiration.
Numerous small metabolite exchange systems are able to
shuttle redox equivalents rapidly across chloroplast and
mitochondrial membranes and thus link NAD(P) pools in the
different compartments (Raghavendra and Padmasree, 2003;
Scheibe et al., 2005). These involve triose phosphate/3-
phosphoglycerate, malate/oxaloacetate, and malate/aspartate
exchange (Day and Wiskich, 1981; Journet et al., 1981;
Heineke et al., 1991). Glutamate/2-oxoglutarate shuttle
systems, which are key in nitrogen assimilation and
ammonia reassimilation (Woo et al., 1987), also potentially
involve exchange of redox equiva-lents. Exchange systems
also operate to link peroxisomal and cytosol NAD pools
(Reumann et al., 1994). As well as indirect redox transfer
between the mitochondrial matrix and the cytosol, evidence
has recently been presented that a cytosolic glycerol-3-
phosphate dehydrogenase (G3PDH) operates with an
externally oriented mitochondrial inner membrane
G3PDH:ubiquinone reductase to deliver elec-trons to the
Arabidopsis mitochondrial electron transport chain, and this
system appears to contribute significantly to cellular redox
homeostasis (Shen et al., 2006). Other externally oriented
alternative dehydrogenases located in the inner
mitochondrial membrane may also allow oxidation of
NADH or NADPH produced in the cytosol or
intermembrane space (Giege et al., 2003; Rasmusson et al.,
2004).
In comparison with the redox exchange between com-
partments, little is known about the transport of NAD and
NADP themselves. As discussed above, available data
suggest that the first three steps of the de novo pathway of
NAD synthesis involve production of quinolinate in the
chloroplast and NaMN formation in the cytosol and
mitochondrion (Fig. 4). In Arabidopsis, only a single gene
1610 Noctor et al.

Fig. 2. Phylogenetic tree and multiple sequence alignment of L-aspartate oxidase (NadB) proteins. (A) The phylogenetic tree was produced from a multiple
sequence alignment using the Clustal W program (http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl). It includes L-aspartate oxidase protein
sequences from Arabidopsis thaliana (AtNadB: GenBank accession number NP_568304), Oryza sativa (OsNadB: XP_464033), Populus trichocarpa (PtNadB:
eugene3.00011954; http://genome.jpgi.pso.org/Potr1/), Synechocystis PCC6803 (SyNadB: NP_442857), E. coli (EcNadB: NP_754979), B. subtilis (BsNadB:
NP_390665), and single homologous sequences extracted from Chlamydomonas reinhardtii

is predicted to encode each of the enzymes necessary for the
subsequent adenylylation and amidation steps. Neither
sequence encodes an apparent transit peptide sequence. In
mammals, three N(a)MNAT isoforms have been character-
ized: one is found in the nucleus, another outside the nucleus
in an undefined location, and the third in both the cytosol
and mitochondria (Magni et al., 2004). A green fluorescent
protein (GFP)yeast NAD synthetase fusion construct
showed a diffuse localization in both the cytosol and the
nucleus (Suda et al., 2003). Taken together, available data
suggest that neither plastids nor mitochondria are in-volved
in the last two steps of NAD synthesis, though this remains
to be confirmed (Fig. 4). Based on this view, there would
appear to be a minimum requirement for plastid inner
membrane transporters able to export quinolinate and import
NAD, as well as for mitochondrial import of NAD and
quinolinate for mitochondrial QPRT activity (Sinclair et al.,
+ +
2000). Both NAD and NADP have been shown to cross
plant mitochondrial membranes (Neuburger and Douce,
1983; Bykova and Mller, 2001), but very little genetic and
molecular information is available on NAD transporters.
As they are relatively large, phosphorylated metabolites,
pyridine nucleotides are usually considered to be exclu-
sively intracellular, with virtually no movement between
cells. However, mammalian plasma contains low concen-
trations of NAD, which are important in signalling through
membrane-bound ADP-ribosyl cyclases such as CD38 that
could act both to produce cADP-R on the extracellular face
of the membrane and to transport it into the cell (Evans et
al., 2005). Recently, a plasma membrane NAD trans-porter
has been shown to operate in a pathogenic in-tracellular
bacterium (Haferkamp et al., 2004). This strain is unable to
synthesize NAD and instead takes it up from the host cell,
probably in exchange for ADP (Haferkamp et al., 2004).
Other bacteria, such as Salmonella typhimu-rium, are able to
take up exogenous NMN via the product of the pnuC gene,
+ nicotine is translocated from roots to shoots, and root
particularly under conditions of low intracellular NAD
(Zhu et al., 1991; Penfound and Foster, 1999), where uptake
activity could be stimulated by the regulatory protein NadR
(see below). Very recently, two genes encoding
+ (Holfelder et al., 1998), and trigonelline probably moves
mitochondrial NAD transporters have been identified in
yeast where, as in plants, the final steps of NAD synthesis
appear to occur outside the mitochondria (Todisco et al.,
2006). Biochemical characterization of one of the gene
+ or organs under physiological conditions is unclear.
products, NDT1, showed high specificity for NAD (Km =
0.38 mM), with virtually no activity
NAD(P) synthesis and pyridine nucleotide cycling 1611
+
with NADH, NADP , or NADPH, but some activity with
other nucleotides, including NaAD (Todisco et al., 2006).
The protein appears to catalyse unidirectional transport at
low rates and exchange with other nucleotides at faster
rates (Todisco et al., 2006). Genome searches reveal that
homologues of the two yeast mitochondrial transporters
exist in taxonomically diverse organisms, including
Arabi-dopsis, poplar, and rice (Fig. 5). The two
Arabidopsis proteins have 3031% identity with yeast
NDT1 and the sequences are currently annotated adenine
nucleotide translocator/mitochondrial substrate carrier,
with a pre-dicted inner mitochondrial membrane location.
It remains to be seen whether similar proteins exist in the
plastid en-velope, and whether mitochondrial import of
NAD occurs in exchange for other nucleotides such as
NaMN, whose fate after production by mitochondrial
QPRT is unclear (Fig. 4). Since NAD(H) kinases appear
to exist in multiple compartments (Gallais et al., 2000a;
Turner et al., 2004, 2005; Chai et al., 2005), the
requirement for NADP transport is yet to be established.
As well as mitochondrial and chloroplast transporters,
important questions remain concerning nuclear NAD
metabolism and nicotinamide recycling, and their
relation-ship to cytoplasmic processes. Imaging analysis
of NAD(H) suggests that the free nuclear NAD :NADH
+
ratio is similar to cytosolic values (Zhang et al., 2002). If
pyridine nucleotides pass readily through nuclear mem-
brane pores, then changes in cellular NAD redox state or
total NAD pools could impact directly on transcription in
animal and yeast cells via redox regulation of the co-
repressor CtBP (Zhang et al., 2002) or substrate availabil-
ity for Sir2 and related proteins (Imai et al., 2000; Lin et
al., 2000). Interestingly, the last two enzymes of NAD
synthesis are present in yeast and mammalian nuclei
(Suda et al., 2003; Magni et al., 2004), and significant
resynthesis of NAD could occur within this compartment.
Intercellular transport is another issue. In tobacco,
synthesis and shoot accumulation are stimulated by stresses
such as wounding (Cane et al., 2005, and references cited
therein). Ricinine is found in the phloem sap of castor bean
between organs in developing mungbean seedlings (Zheng et
al., 2005). Whether any of the other intermediates and
precursors shown in Fig. 1 move significantly between cells

(CrNadB: http://chlamy.org) and Physcomitrella patens (PpNadB: http://moss.nibb.ac.jp) databases by BLAST search against the other plant and bacterial
sequences shown. A homologous sequence from Medicago truncatula (MtNadB: BAC accession number AC159962, http://www. medicago.org) was also
included in the analysis. Gaps were excluded. The numbers indicate percentage identities between the protein sequences. (B) Sequence alignment of L-
aspartate oxidase proteins. Identical amino acids are highlighted in black and include residues shown for the E. coli enzyme to
be involved in FAD (S22, A25, K44, Q56, G58, A164, and G382) and succinate (G223, E268, R298, H361, and S397) binding, numbered for the Arabidopsis protein.
Sequences in frames indicate similarity. Transit peptides of plant and Chlamydomonas sequences predicted by ChloroP software (http://www.
cbs.dtu.dk/services/ChloroP/) were not taken into account in the alignment.
1612 Noctor et al.

Fig. 3. Phylogenetic tree and multiple sequence alignment of quinolinate synthase (NadA) proteins. (A) The tree was produced as described for Fig.
2 by comparing quinolinate synthases from A. thaliana (AtNadA: GenBank accession number NP_199832), O. sativa (OsNadA: ABA_97161), P.
trichocarpa (PtNadA: eugene3.00150643; http://genome.jpgi.pso.org/Potr1/), M. truncatula (MtNadA: sequence number TC107403 from the TIGR
database; http://www.tigr.org/tdb/tgi/plant.shtml), P. patens (PpNadA: http://moss.nibb.ac.jp), C. reinhardtii (CrNadA: AAK_54345), Syne-chocystis
PCC6803 (SyNadA: NP_442873), E. coli (EcNadA: NP_752755), and B. subtilis (BsNadA: NP_390663). Gaps were excluded. The numbers
indicate percentage identities between proteins. (B) Sequence alignment of various quinolinate synthase proteins. Identical amino acids are
highlighted in black. Sequences in frames indicate similarity. Because the available Chlamydomonas sequence is incomplete in the C-terminal
domain, only parts of the sequences are aligned.

Stress-induced changes in NAD status
Given the centrality of pyridine nucleotides to cell function,
it seems likely that contents are highly regulated. Operation
of NAD-consuming reactions means that regulation will be
necessary to ensure homeostatic replenishment of NAD
pools. Furthermore, adjustment of NAD pools could play an
important part in environmentally induced dynamic
signalling, in which the cell is able to sense and respond to
changes in concentrations of NAD and/or precursors or
metabolites (Zhang et al., 2002).
Several NAD-consuming reactions could be important in
stress conditions and/or signalling in interactions with ROS
and other redox components. Most of these reactions release
nicotinamide, which can be recycled to NAD by a salvage
pathway (Fig. 1). One important NAD-consuming enzyme
induced by stress is PARP. This nuclear enzyme transfers
+ as quinolinate or nicotinamide, which are known to be
ADP-ribose units from NAD to residues on
target proteins, including itself, releasing nicotinamide
and ultimately forming long poly(ADP-ribose) chains that
are important in regulating processes such as DNA repair
(Berglund et al., 1996; Graziani et al., 2005). Hyper-
activation of PARP-1 by ROS or nitric oxide has been
shown to deplete NAD pools in mammalian and plant
cells, and decreasing PARP inhibition can diminish death
in both types of cell (Heller et al., 1995; Amor et al.,
1998). Recently, it has been shown that stress-induced
+
PARP activation causes NAD and ATP depletion, and
that these effects and the attendant cell death are atten-
uated in lines in which PARP expression is diminished by
RNA interference (RNAi) (De Block et al., 2005).
As well as producing changes in NAD availability,
adjustments in NAD metabolism could impact on stress
signalling through changes in metabolic intermediates such

Fig. 4. Possible subcellular compartmentation of the de novo NAD(P)
synthesis pathway. Established or likely enzymatic conversions are
indicated by solid arrows, uncertain reactions by dashed arrows, and
transport steps by dotted arrows. Other pathways that are not shown could
also be important (see text). Only transport steps that seem to be necessary
based on likely compartmentation of synthetic enzymes are considered.
Other transport reactions could also occur (e.g. transport of NaAD).
Localization of synthetic enzymes is based on available gene sequences, as
well as the reported localization of quinolinate synthesis enzymes in the
chloroplast (Katoh et al., 2005). A tobacco cDNA for quinolinate
phosphoribosyltransferase (QPRT) is predicted to encode a protein targeted
to the mitochondria, although a second transcript may also be produced
from the same gene to produce a shorter protein (Sinclair et al., 2000). The
Arabidopsis gene predicts two proteins, one lacking the putative
mitochondrial target sequence, suggesting that QPRT is found in both the
mitochondria and the cytosol. The subsequent steps of NAD synthesis are
probably located in the cytosol, although other locations cannot yet be
definitively discounted. NADP synthesis occurs in the chloroplast and the
cytosol, and, perhaps, in the mitochondria (Gallais et al., 2000a; Turner et
al., 2004, 2005; Chai et al., 2005). Mitochondria can import NAD and
NADP (Neuburger and Douce, 1983; Bykova and Mller, 2001). For the
transporter activities indicated by ellipses, the only identified protein is the
yeast mitochondrial NDT transporter (Todisco et al., 2006), for which
homologous sequences exist in Arabidopsis, poplar, and rice (see Fig. 5).
NDTh, homologue of the yeast NDT. Other abbreviations as for Fig. 1.
neuroactive compounds in animals (Stone and Addae, 2002;
Magni et al., 2004). Quinolinate accumulation in the brain
has been implicated in several neurodegenerative disorders
(Fukuoka et al., 2002, and references therein). Nicotinamide
is known to be an inhibitor of nicotinamide-generating
enzymes such as PARP and Sir2 (Magni et al., 2004; De
Block et al., 2005), and high concentrations have been
shown to influence defence processes in plants (Berglund et
al., 1993, 1996). Besides PARP, other en-zymes can modify
the biological activity of proteins by transferring a single
ADP-ribose moiety from NAD to target amino acids. An
Arabidopsis mutant, rcd1, which shows hypersensitive
responses to extracellular super-oxide, contains a PARP-type
NAD-binding signature that could reflect the mono(ADP-
ribose) transferase function (Ahlfors et al., 2004).
Several literature studies show that NAD pools are plastic
in plants, and can undergo significant changes in response to
the environment. Powdery mildew infection of barley leaves
was reported to be associated with increased
NAD(P) synthesis and pyridine nucleotide cycling 1613
NAD contents, although no evidence was obtained for
increases in synthesis (Ryrie and Scott, 1969). It is evident
from Fig. 1 that NAD contents could be modified by
changes at multiple levels, including degradation and re-
cycling efficiency, as well as de novo synthesis. Within this
context, key points relate to the removal of metabolites from
the recycling pathway for pyridine alkaloid synthesis,
degradation of nicotinamide, or formation of nicotinate or
nicotinamide conjugates (Fig. 1). Enzymes able to
decarboxylate and oxidize both pyridine monocarboxylates
(nicotinate, isonicotinate, and picolinate) and dicarboxy-lates
(quinolinate and dipicolinate) to hydroxylated prod-ucts are
found in bacteria (Uchida et al., 2003). Another question
concerns stress-induced effects on the NAD: NADP ratio. In
cultured tobacco cells expressing a hyper-active calmodulin,
increased activity of NAD kinase, a calmodulin-activated
enzyme, was associated with a sig-nificant increase in the
+
NADP :NAD ratio (Harding et al., 1997). Interestingly,
NAD kinase from Bacillus subtilis is allosterically activated
by quinolinate (Garavaglia et al., 2003), and this could play
some role in the co-regulation of the de novo synthesis of
NAD and NADP.
Further evidence pointing to a role for the concentration
of NAD and/or associated metabolites in influencing stress
resistance comes from analysis of tobacco lines deficient in
mitochondrial complex I. These plants respire through
alternative respiratory dehydrogenases. Growth is slowed
but leaf respiration is increased, as is resistance to both
abiotic and biotic stress (Dutilleul et al., 2003a, b). Enhanced
resistance is accompanied by a 2-fold increase in leaf
+
contents of NAD and NADH, without an appre-ciable
+
increase in NADP , NADPH, H2O2, ascorbate, or
glutathione (Dutilleul et al., 2003b, 2005). From a physio-
logical point of view, increases in NADH in these plants can
perhaps be rationalized in terms of the relatively high K m
NADH of the alternative matrix-orientated mito-chondrial
NADH dehydrogenase, which in this line is likely to be
important as a functional replacement of complex I. The
biochemical and molecular processes re-sponsible for
increases in NAD in the cytoplasmic male sterile (CMS)
mutant remain unidentified, and further work is required to
determine the extent to which the observed changes in NAD
status in these plants contribute to their enhanced resistance
to stress (Dutilleul et al., 2003b).
The extent to which the redox state of NAD and NADP
pools changes in response to the environment is still a matter
of some debate. Concerning the chloroplast, it is often
assumed that increasing light causes progressive over-
reduction at the reducing side of photosystem I (PSI),
+
decreasing the availability of NADP , the principal oxidant
of ferredoxin, and so favouring diversion of electrons to
oxygen to produce ROS. However, the stromal redox state is
rather constant in response to changes in irradiance, though
less so when metabolism strongly limits electron transport,
for example, at low temperatures or if both CO2
1614 Noctor et al.

+
Fig. 5. Phylogenetic tree and multiple sequence alignment of yeast mitochondrial NAD transporters NDT1 and NDT2 (Todisco et al., 2006) and homologous
protein sequences in other organisms. (A) The tree was produced as described for Figs 2 and 3, by BLAST search with Saccharomyces cerevisiae NDT1 as
probe. (B) Sequence alignment of yeast NDTs and homologous sequences in other organisms. Identical amino acids are highlighted in black. Sequences in
frames indicate similarity. Key to sequences: ScNDT1, Saccharomyces cerevisiae NP_012260; ScNDT2, S. cerevisiae
 NAD(P) synthesis and pyridine nucleotide cycling 1615

and O2 are artificially decreased (Harbinson et al., 1990).
Direct measurements of fractionated pea leaf protoplasts
showed that cytosolic NADP redox states were relatively
independent of light, dark, or CO2 availability (Igamberdiev
and Gardestro m, 2003). However, cytosolic NAD and both
mitochondrial NAD and NADP pools were more reduced in
the light, particularly under conditions favouring
photorespiration (Igamberdiev and Gardestro m, 2003). It
was proposed that these changes are an important factor in
mediating light-induced redirection of leaf metabolism,
particularly via inhibition of the mitochondrial NAD-ICDH
caused by more reduced NAD pools (Igamberdiev and
Gardestro m, 2003). Such phenomena could be at least
partly responsible for some of the changes in metabolite
profiles observed in the tobacco CMS mutant, where
+ grouped in the nad operon under the control of a single
increases in total leaf NAD and NADH are associated with
enhanced nitrate reduction and an increased citrate:2-
oxoglutarate ratio despite enhanced extractable NAD-ICDH
activity (Dutilleul et al., 2005). However, as for almost all
other similar work, pyridine nucleotide measure-ments in
these studies represent total nucleotide pools, i.e. they
include nucleotides that are bound to proteins as well as the
more biochemically relevant free pools. Recent analyses of
isolated potato tuber mitochondria in short-term experiments
have led to the conclusion that free mitochon-drial NADH is
+ been described. First, as well as the DNA-binding repressor
maintained at a more constant value than total NAD and
NAD, at least in organelles isolated from non-photosynthetic
cells (Kasimova et al., 2006). Several other recent studies
using leaf systems indicate that changes in cytosolic and
mitochondrial NAD status are important in the lightdark
regulation of nitrate reduction and re-spiratory pathways
associated with nitrogen assimilation (Kaiser et al., 2002;
Igamberdiev and Gardestro m, 2003; Rachmilevitch et al.,
2004; Dutilleul et al., 2005).
Notwithstanding the attention paid to chloroplastic
events using non-invasive techniques such as chlorophyll
fluorescence, the impact of stress on NAD(P) pools and
redox states in the different compartments remains un-
resolved. NAD(P)H oxidases are among the key players
in ROS signalling, and PARP (and perhaps other NAD-
consuming enzymes) can be activated by stress. In this
context, key questions relate, first, to possible links be-
tween NAD degradation/(re)synthesis and redox states of
NAD(P) and, secondly, to what extent NAD(P) pools in
different compartments are autonomous or interdepend-
ent. Given that NAD(P)H oxidases use cytosolic re-
ductant and several NAD-consuming enzymes are located
in the nucleus, the cytosol and nucleus could be the key
locations for NAD-linked signalling. Further information
is required to establish whether chloroplast and mitochon-
drial pools might be relatively insulated from potentially
larger fluctuations in cytosolic NAD. Within this context,
the location of the different reactions of the pyridine
cycling pathway is an important consideration.
Regulation of NAD contents
What are the mechanisms that could act to regulate NAD
synthesis in plants? In bacteria, in which NAD metabolism
involves similar enzyme activities to those found in plants,
regulation occurs at transcriptional and post-translational
levels. The bacterial nadA, nadB, and nadC genes are
promoter (Penfound and Foster, 1996). In S. typhimurium,
transcription of nadA, nadB, and pncB (encoding NaPRT) is
repressed by the DNA-binding function of the repres-sor
protein NadR (Penfound and Foster, 1999). Binding of NadR
+
to the nadA operator is highly dependent on NAD , and
uninfluenced by physiological concentrations of qui-
nolinate, nicotinate, nicotinamide, NaMN, NMN, NaAD,
+
NADH, or NADP (Penfound and Foster, 1999). NadR is an
intriguing protein for which two additional activities have
+
function that is favoured in NAD -replete cells, NadR could
+
act in NAD -deficient cells as a transport facilitator in
conjunction with the pnuC gene product, encoding a NMN
transporter protein (Penfound and Foster, 1999). Secondly,
in addition to repressor and transport functions, NadR also
has dual nicotinamide riboside kinase/NMNAT activity.
These activities have been described for NadR from several
bacteria, but seem to be particularly important in Haemo-
philus influenzae. In this species, which lacks the nadA,
nadB, nadC, and pncB genes and thus many of the enzymes
shown in Fig. 1, the DNA-binding domain is absent from
NadR, but the presence of the enzymatic domains enables
+
NAD to be produced from absorbed nicotinamide riboside
(Singh et al., 2002). In B. subtilis, no homologues of NadR
are found, but another repressor, YrxA, regulates nadA and
nadB transcription (Rossolillo et al., 2005). In this
bacterium, however, the quinolinate synthesis path-way is
regulated antagonistically with the pyridine cycling pathway
because the metabolite that enhances the DNA-binding
activity of YrxA is nicotinate, not NAD, and a further
difference from the NadR system is

NP_010910; AtA, ScNDT1 homologue A from A. thaliana, protein NP_564233, gene At1g25380; AtB, ScNDT1 homologue B from A. thaliana,
protein NP_566102, gene At2g47490; PtA, ScNDT1 homologue A from P. trichocarpa, fgenesh1_pm.C_LG_X000398; PtB, ScNDT1 homologue B
from P. trichocarpa, eugene3.00140704; OsA, ScNDT1 homologue A from O. sativa, protein BAD73272 (currently annotated as a folate transporter,
gene identifier OS01G32980); OsB, Sc NDT1 homologue B from O. sativa, protein AAV43843, gene OS05G28870 (currently annotated as a
mitochondrial carrier protein); Hs, Homo sapiens protein NP_110407 (currently annotated as a folate transporter); Xl, Xenopus laevis protein
AAH87370; DrA, ScNDT1 homologue A from Danio rerio (Zebrafish), protein AAH90770; DrB, ScNDT1 homologue B from D. rerio, protein
NP_956550; and Dm, ScNDT1 homologue from Drosophila melanogaster, protein AAM68821.
1616 Noctor et al.
that YrxA does not repress pncB transcription (Rossolillo
et al., 2005). The end-result is that quinolinate synthesis is
only activated in B. subtilis when nicotinate is not avail-
able. Analysis of available sequences suggests that no
NadR or YrxA homologues are to be found in plants, and
the importance of transcriptional control of NAD
synthesis is unclear.
+
In E. coli, NAD and NADH concentrations can post-
translationally regulate NAD synthesis by feedback in-
hibition. An in vitro assay of the E. coli nadA and nadB gene
products in combination showed that NAD concen-trations
close to physiological (13 mM) strongly inhibited
quinolinate formation, while NADP had much less effect
(Griffith et al., 1975). It was subsequently shown that the
+
first enzyme, L-aspartate oxidase, is inhibited by NAD
(competitively with the prosthetic group FAD), while the
second enzyme, quinolinate synthase, is inhibited by both
+
NAD and NADH (Nasu et al., 1982b). Thus, decreases in
+
NAD should release feedback inhibition over quinoli-nate
synthesis. If the plant homologues are subject to similar
regulation, a key point will concern the relationship between
NAD status outside and within the chloroplast, where
quinolinate synthesis is probably located (Fig. 4). It is
possible that the most important adjustments of quin-olinate
synthesis could occur through changes in protein
concentrations, mediated by transcriptional or post-
transcriptional processes that result from changes in cyto-
+ an example of differential regu-lation of the NAD synthesis
solic or nuclear NAD concentration, NAD :NADH ratios, or
other signal. Quinolinate or nicotinate could also have some
direct inhibitory effect on the enzyme activities.
Post-translational mechanisms acting through covalent
modification of enzymes or through proteinprotein inter-
actions are also likely to be important in regulating the web
of reactions shown in Fig. 1. Certain plant NAD kinases are
regulated by calmodulin (Harding et al., 1997) and also
perhaps by thioldisulphide exchange (Delumeau et al.,
2000). An important issue is the extent to which quinolinate
synthesis in plants requires complex formation between L-
aspartate oxidase and quinolinate synthase. Reversible complex
formation between enzymes is known to be a key mode of
regulation of plant metabolic pathways such as cysteine
synthesis from sulphide and serine (Droux, 2004).
An important question concerns the importance of
quinolinate synthesis compared with nicotinamide recyc-ling
in different plant tissues or environmental conditions, or at
specific stages of development. Labelling studies suggest
that NAD and NADP pools turn over within a few hours
even under optimal conditions (Ryrie and Scott, 1969).
Turnover times could be significantly shortened by increased
NAD utilization, and in these conditions the pyridine
recycling path may be particularly important. NaPRT is
considered to be the limiting enzyme in the recycling
pathway in bacteria, and overexpression of this enzyme in E.
coli can cause a 5-fold increase in cellular NAD
concentrations in the presence of added nicotinate
(Wubbolts et al., 1990). More recently, it was shown that
expression of the S. typhimurium enzyme in E. coli
increased NAD pools ;1.5-fold and that this was associ-ated
+
with increased NAD :NADH ratios (Berros-Rivera et al.,
2002). In barley leaf tissue, NAD was labelled at similar
14 14
rates from [ C]nicotinate and [ C]quinolinate (Ryrie and
Scott, 1969). It might be predicted that rapidly growing
tissue would require an active de novo synthesis from
quinolinate whereas the recycling pathway could perhaps be
more important in mature tissues such as fully expanded
leaves. In theory, the de novo synthesis pathway could be
dispensable in expanded cells if recycling is close to 100%
efficient. One obvious factor that would affect recycling
efficiency is the extent of pyridine leakage from the
pathway, for instance via pyridine ring degradation.
A second factor impacting on pyridine cycling is the use
of pyridine rings to produce compounds other than
nucleotides. Regulation in such systems probably differs
from that operating in cells in which the primary pyridine
compounds are NAD and NADP. Tissue NAD and NADP
pools in plants are relatively small, typically of the order of
71
20100 nmol g fresh weight (Dutilleul et al., 2005; Shen et
al., 2006). By contrast, pyridine alkaloids can ac-cumulate
much more strongly in certain species (Mann and Byerrum,
1974b). Trigonelline contents in coffee leaves can reach 20
71
lmol g fresh weight (Zheng et al., 2004). Tobacco presents
pathway between tissues that produce nicotine and those that
do not. Analysis of QPRT in Nicotiana species that
predominantly produce nicotine (N. tabacum and N.
sylvestris) showed that transcripts were confined to roots
(where nicotine is synthesized) while QPRT was expressed
in both roots and leaves in N. glauca, in which the principal
pyridine alkaloid is anabasine (Sinclair et al., 2000).
Wounding of aerial tissues greatly increased QPRT
transcripts in all three species, although it is notable that
induction was confined to the roots in the nicotine-producing
species (Sinclair et al., 2000). Pre-liminary data on L-
aspartate oxidase and quinolinate synthase also appear to
suggest that, in tobacco, the de novo pathway of NaMN
synthesis is most highly expressed in conditions in which
nicotine synthesis is stimulated. Like QPRT, these enzymes
appear to be most expressed in the tobacco root and are
similarly induced by methyl jasmonate (Katoh and
Hashimoto, 2004). In contrast, they are not induced by
jasmonate treatment of tobacco leaves (Katoh and
Hashimoto, 2004). In castor bean seedlings accumu-lating
ricinine, extractable QPRT activities were much higher than
those of NaPRT, but in mature leaves, in which ricinine
synthesis is much less significant, the two activities were
similar (Mann and Byerrum, 1974b). The same study
reported that of the two enzymes in tobacco, QPRT was the
more active, whereas in other species the two activ-ities
were comparable (Mann and Byerrum, 1974b). High or
increased activity of quinolinate production and

use presumably reflects enhanced production of alkaloids
as feeding deterrents. Whether the de novo pathway of
NAD synthesis has any role to play in more general
conditions of stress, in which NAD metabolism is stimu-
lated or perturbed, remains to be seen.
Conclusions and perspectives
Work in animal and yeast systems on the signalling roles of
NAD and derivatives has rekindled interest in pyridine
nucleotide synthesis and turnover. Recent studies in plants
suggest that NAD contents are both flexible and potentially
important in determining cell fate. These developments are
introducing a new dimension into studies of pyridine
nucleotides, in which studies of synthesis and turnover are
examined alongside the hitherto dominant analysis of redox
state and redox flux. In relation to stress physiology, a key
outstanding question is the extent to which plants adjust their
NAD pools in different conditions. Linked to this issue is the
eludication of the roles of enzymes such as PARPs,
SIRTUINS, and cADP-R in plants, and the impact of these
activities on NAD contents, turnover, and synthesis. This
review has focused specifically on the pro-duction and
metabolism of the pyridine ring of NAD(P), but it is clear
that future work on NAD will also have to consider co-
ordination of purine and pyridine nucleo-tide metabolism. As
well as representing the major product of NAD(P)H
oxidation in biological energy transduction, ATP is also
required to produce the adenosine mono-phosphate moiety
of NAD(P) and as a phosphate donor in several reactions
shown in Fig. 1.
Together, molecular, genetic and biochemical studies are
beginning to elucidate the web of reactions underpinning
NAD synthesis and pyridine recycling in plants. Current
knowledge strongly suggests that like bacteria, plants make
quinolinate from aspartate and triose phosphate. Genes
enabling quinolinate synthesis from tryptophan appear to be
absent from plant and Chlamydomonas genomes. Certain
pathogenic bacteria have been identified that use the
tryptophan pathway (Kurnasov et al., 2003), but no organism
has been unambiguously identified that uses both routes for
quinolinate synthesis. Taken together, the genomic data
suggest that there is no simple schism be-tween prokaryotic
and eukaryotic organisms with respect to quinolinate
synthesis. It is intriguing that enzymes catalysing the two
steps seem to be each encoded by a unique gene and that
both sequences are relatively highly conserved between
divergent taxonomic groups. This suggests a strong selection
pressure on this pathway, re-flecting its fundamental
importance in plant and bacterial cell life. Indeed, the
pathway has been proposed as an example of how abiotic
chemical evolution may have occurred in pre-cellular
systems (Wachtershauser, 1988). One element of this
engaging theory is chemical auto-catalysis through
mechanisms that are independent of
NAD(P) synthesis and pyridine nucleotide cycling 1617
polypeptide enzymes, and it is suggested that quinolinate
formation could have been autocatalytically stimulated by
NaMN-dependent oxidation of aspartate to iminoaspar-
tate (Wachtershauser, 1988). Following the evolution of
enzyme catalysts, the flavoprotein L-aspartate oxidase (or
precursor) would have taken over this role.
The plant pyridine cycling pathway seems to have more
in common with the bacterial pathway than with animal and
fungal models. Additional reactions could also be present in
plants, enabling enzymes to be by-passed under certain
conditions and hence conferring potentially important
metabolic flexibility. Reverse genetics approaches will be
necessary to establish the necessity or otherwise of such
reactions. The question of enzyme specificity is also key.
Biochemical characterization of the purified enzymes will be
required to confirm the putative preferences of the different
phosphoribosyltransferases and adenylyltrans-ferases. It
remains to be seen whether the NaMN adenylyl-transferase
(a single annotated gene in Arabidopsis) has any role in
direct production of NAD from NMN. Metabolite profiling
could also provide useful comp-lementary information on
the presence of given reactions in plant tissues, and
understanding the integration of NAD(P) synthesis and
turnover will require confirmation of the subcellular
localization of enzymes and character-ization of intracellular
transport systems.
Acknowledgements
We thank Vincent Thareau for expert advice on gene sequence
analysis, and an anonymous reviewer for constructive comments
that helped to improve the manuscript in revision.
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