Download Purine Biosynthesis. Big in Cell Division, Even

Update on Purine Biosynthesis
Purine Biosynthesis. Big in Cell Division, Even Bigger in
Nitrogen Assimilation
Penelope M.C. Smith* and Craig A. Atkins
Botany Department, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia
6009, Australia
Synthesis of the purine ring is a central metabolic
function of all cells. The products, AMP and GMP,
provide purine bases for DNA and RNA, as well as
for a number of essential coenzymes (NAD, NADP,
FAD, and coenzyme A) and signaling molecules (e.g.
cAMP; Fig. 1). ATP serves as the energy source for
many chemical reactions. In addition, in plants, the
nucleotides are the precursors for purine alkaloids,
and for the adenine moiety of cytokinin plant growth
regulators (Fig. 1). Despite the essential functions for
purines, salvage pathways, which retrieve the purine
ring after nucleic acid or coenzyme breakdown, recycle nucleotides to meet day-to-day needs. Thus, the
requirement to synthesize new purines in differentiated cells is small. It is only when DNA is replicated
that de novo synthesis of the purine ring is required
and so, although present in most tissues, the activity
of the metabolic pathway is relatively low.
PURINE SYNTHESIS AS A PATHWAY
FOR N ASSIMILATION?
The purine pathway also functions in pathways
that are different and distinct from these “housekeeping roles.” It is employed in specialized tissues to
assimilate and detoxify NH3. In the liver of birds and
terrestrial reptiles, purines synthesized de novo are
oxidized to uric acid and excreted as the main form
of waste N. Plants do not engage in N excretion but
rather store it. In members of the Aceraceae, Plantanaceae, and Borraginaceae, N storage is accomplished
as purines are oxidized to ureides (allantoin and
allantoic acid). In such plants, ureides are the dominant forms of stored N in stems and underground
organs (Reinbothe and Mothes, 1962), accounting for
as much as one-half the plant’s N in some species,
such as comfrey (Symphytum offinalis). However, it is
in nodules of tropical legumes, such as soybean (Glycine max) and cowpea (Vigna unguiculata), that the
pathway plays a dominant role in primary N metabolism (Atkins and Smith, 2000; Fig. 1). Within cells
infected with rhizobia, bacterial nitrogenase activity
leads to secretion of fixed N, principally as NH3 or
NH4⫹ (Fig. 2). Although assimilated initially through
* Corresponding author; e-mail [email protected];
fax 61– 08 –9380 –1001.
www.plantphysiol.org/cgi/doi/10.1104/pp.010912.
the amide group of Gln (by Gln synthetase), almost
all fixed N is subsequently incorporated through the
purine pathway to form IMP, and finally ureides. In
these species, ureides are translocated in xylem from
nodules and provide the major supply of N for the
plant’s nutrition (Fig. 2). To accommodate this flux of
fixed N in nodules, activity of enzymes in the purine
pathway is enhanced considerably compared with
other tissues, including active meristems (Atkins and
Smith, 2000). For this reason, nodules have been exploited as the tissue of choice in which to study the
enzymology of purine biosynthesis in plants.
A SELECT FEW LEGUMES CHOOSE THE
PURINE PATHWAY
The formation and translocation of fixed N as ureides is not a feature of all nodulated legumes but is
restricted, almost exclusively, to species of the tribes
Phaseoleae, Desmodieae, and Indigofereae (Atkins,
1991) within the Phaseoloid group (Doyle et al.,
2000). Species in the Phaseoleae and Desmodieae
form “desmodioid” nodules. These are typically
more or less spherical; as for soybean or cowpea, they
develop from infection through root hairs, have a
central infected tissue zone that is interspersed with
uninfected cells (Fig. 2), and are determinate (Corby,
1988). However, desmodioid nodules are also a feature of the Loteae, a temperate tribe that is taxonomically distant from the Phaseoloid group (Doyle et al.,
2000) and does not assimilate fixed N as ureides.
Among the Indigofereae, only Cyamopsis tetragonoloba
has been reported to translocate significant levels of
ureide in xylem (Atkins, 1991) and its nodules are not
desmodioid, but indeterminate.
The ureide pathway occurs in determinant nodules
where the N-fixing tissue zone includes both infected
and uninfected cells (Fig. 2), an arrangement that
apparently facilitates the function of uricase, the enzyme catalyzing the ultimate step of purine oxidation
(Fig. 1). Uricase requires molecular O2, but has an
apparent Km (O2) that is extremely unfavorable (30
␮m; Rainbird and Atkins, 1981) in nodules where the
average pO2 in infected cells is maintained at 10 to 60
nm in solution (Kuzma et al., 1993). Thus, it is not
surprising that uricase expression is confined to
greatly enlarged microbodies in interstitial, uninfected cells of the N-fixing zone (Fig. 2). Here, pO2 is
Plant Physiology, March 2002, Vol.Downloaded
128, pp. 793–802,
from on
www.plantphysiol.org
June 16, 2017 - Published
© 2002
by American
www.plantphysiol.org
Society of Plant Biologists
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
793
Smith and Atkins
Figure 1. Schematic diagram for the pathway of de novo purine biosynthesis (the box at the top of the figure) and further
metabolism of purines in plant cells. The first committed step of the purine pathway, catalyzed by phosphoribosylpyrophosphate amidotransferase (PRAT), is one of a number of possible metabolic fates for both substrates (phosphoribosylpyrophosphate [PRPP] and Gln [gln]). The final product of the pathway, inosine monophosphate (IMP), is the first purine
nucleotide and provides the basic purine ring structure for the other purine nucleotides, xanthosine monophosphate (XMP),
(Legend continues on facing page.)
794
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Purine Biosynthesis
Figure 2. Two types of cell in the central infected tissue zone of nodules are required for the synthesis of ureides from fixed
N. The enlarged infected cells (IC) contain as many as 50,000 rhizobia (Bact), differentiated into bacteroids and enclosed
in groups within membrane (peribacteroid membrane) vesicles of plant origin. These bacteria express nitrogenase and export
ammonia to the host cell cytosol where it is assimilated as the amide group of Gln (gln). Gln, together with other amino
acids, is used by both plastids (P) and mitochondria (M) to form purines. These plant organelles are localized at the periphery
of the infected cell and are concentrated particularly adjacent to gas spaces. The purines are oxidized within the infected
cell cytosol to urate that is transferred to uninfected cells (UC) of the central zone and further oxidized within enlarged
microbodies (Mb) to allantoin and allantoic acid (ureides). Ureides are exported from the nodule in xylem to provide the
majority of the N for the plant’s nutrition.
Figure 1. (Legend continued from facing page.)
AMP, and GMP. The last two of these are the building blocks of DNA and RNA, whereas AMP is the precursor for the
cytokinin group of plant growth regulators and a number of important coenzymes. GTP and ATP participate in the energy
metabolism of the cell. Although there are a number of routes that can generate the purine bases from IMP, in legume nodules
the preferred route is through IMP dehydrogenase (IMPDH). Both xanthosine and xanthine serve as precursors for the purine
alkaloids (theobromine and caffeine) and their further oxidation yields the ureides, allantoin and allantoic acid. Enzyme names
are abbreviated as follows: glycinamide ribonucleotide synthetase (GARS), glycinamide ribonucleotide transformylase (GART),
formylglycinamide ribonucleotide amidotransferase (FGARAT), aminoimidazole ribonucleotide synthetase (AIRS), aminoimidazole ribonucleotide carboxylase (AIRC), succinoaminoimidazolecarboximide ribonucleotide synthetase (SAICARS),
adenylosuccinate-AMP lyase (ASAL), aminoimidazolecarboximide ribonucleotide transformylase (AICART), inosine monophosphate cyclohydrolase (IMPCH), and xanthine dehydrogenase/xanthine oxidase (XDH/XO). Substrate names are abbreviated as follows: phosphoribosylamine (PRA), glycinamide ribonucleotide (GAR), formylglycinamide ribonucleotide
(FGAR), formylglycinamidine ribonucleotide (FGAM), aminoimidazole ribonucleotide (AIR), carboximideaminoimidazole
ribonucleotide (CAIR), succinoaminoimidazolecarboximide ribonucleotide (SAICAR), aminoimidazolecarboximide ribonucleotide (AICAR), formylaminoimidazolecarboximide ribonucleotide (FAICAR), XMP, GMP, Gln (gln), Gly (gly), N10-formyl
tetrahydrofolate (N10-formyl THF), Asp (asp), and inorganic phosphate (Pi). Genes encoding the enzymes of the purine
pathway are shown in parentheses beneath the enzyme abbreviation.
Plant Physiol. Vol. 128, 2002
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
795
Smith and Atkins
predicted to be higher and adequate to support urate
oxidation at rates commensurate with those of N2
fixation (Thumfort et al., 1999).
Species of other tribes in the Papilionoideae assimilate fixed N as Gln and Asn and these, especially
Asn, are translocated out of nodules. Data for the
Caesalpinoideae and Mimosoideae are neither extensive nor well documented (Atkins, 1991). There have
been reports of significant ureide translocation in
species outside the Phaseoloid group (Atkins, 1991),
but a number of these have been because of analytical
artifacts (for references, see Atkins and Smith, 2000).
Similarly, there have been reports of low levels of
ureide in xylem exudate or in extracts of nodules
from species that assimilate fixed N principally as
Asn (Brown and Walsh, 1994). Thus, in Robinia
pseudoacacia, 3% of xylem-N was ureide (Atkins et al.,
1991) and in Lotus japonicus, Medicago sativa, and
Medicago truncatula (Cheng et al., 2000), levels less
than 10% compared with Asn-N have been reported.
In these species, uninfected cells differentiate in the
central zone and express uricase (Atkins et al., 1991;
Tajima et al., 2000). Thus, it seems likely that small
amounts of ureide are formed from nucleic acid
breakdown accompanying senescence, in much the
same way as ureide accumulates transiently in the
cotyledons of germinating seeds (Fujihara and
Yamaguchi, 1978).
AND IT IS NODULE SPECIFIC, TOO!
Ureide synthesis in nodulated legumes has attracted interest in part because many of the species
that show this metabolism are important crops (soybean, cowpea, common bean [Phaseolus vulgaris], and
mung bean [Vigna radiata]), but also because utilization of this pathway for N assimilation is greatly
enhanced in nodules. Roots and other tissues of soybean or cowpea assimilate soil mineral N (NO3⫺ or
NH4⫹) into the amides Gln and Asn (Atkins and
Smith, 2000) and these are the translocated forms of
N in both xylem and phloem. Thus, elevated expression of de novo purine synthesis is a specific metabolic feature of the symbiosis in ureide-forming legumes. In fact, the unique association of ureide
synthesis with nodules has proven sufficiently specific that an assay for xylem-borne N-solutes has
been developed as the basis of a practical field
method to estimate relative proportions of fixed and
soil N in soybean.
WHY ASSIMILATE N AS PURINES?
Purine/ureide synthesis would seem to be an extraordinarily complex mechanism for NH3 assimilation, requiring enhanced expression of 20 separate
enzymes as compared with just four required for Asn
synthesis. Furthermore, two of the steps involve
one-C additions as formyl-tetrahydrofolate deriva796
tives (Fig. 1), requiring enhanced activity of enzymes
for their synthesis also. Thus, it is reasonable to ask:
why purine/ureides in some species and not in others and what advantages/disadvantages might accrue as a result of this trait?
Despite the biochemical complexity of the pathway, the “cost” in terms of ATP and reductant expended per N assimilated is not much different from
that required for Asn. Ureides are made up of four C
and four N atoms (Fig. 1), having a C:N ratio of 1.0
compared with 2.5 and 2.0 for Gln and Asn, respectively. Thus, less C is required for translocation of
fixed N as ureide. Theoretical considerations of the
likely C to N economy of nodules of amide-forming
versus ureide-forming species indicate that this “saving” of translocated C could be significant. An attempt to compare experimentally the C/N economy
of an amide former (white lupine [Lupinus albus])
with a ureide former (cowpea) indicated a significantly lower level of C consumption and loss as CO2
by nodules of the ureide former (Layzell et al., 1979).
However, the two symbioses also differed in levels of
phosphoenolpyruvate carboxylase and rates of H2
evolution as well as the proportional distribution of
infected and cortical tissue. These, as well as ureide
versus amide synthesis, were also likely to affect the
relative C costs of fixation. There are no other data
available that approach this question, and attempts
to isolate mutants of ureide formers that assimilate
fixed N as amides have not been successful. As a
consequence, whether one strategy for NH3 assimilation is more or less costly remains unresolved.
There is a growing body of evidence indicating that
pathways of C and N assimilation are coordinately
regulated and that C to N signaling networks in
plants may be a central feature influencing growth
and development (Coruzzi and Zhou, 2001). However, whether this extends to the “sensing” of the C
to N ratio per se through specific N solutes in sources
and sinks and in translocation pathways that link
them is not known.
Despite the putative economy of C use in translocating N in the form of ureides, ureides are not as
suitable as Asn or Gln as a source of N for amino acid
and protein synthesis. Their metabolism in the shoot
involves release of all N as NH3, with the need for
reassimilation. Similarly, most of the ureide-C is released as CO2. As a consequence, little xylem-borne
ureide is transferred as such to phloem in leaves but
rather its N is reassimilated to Asn and Gln, and
these constitute the principal N solutes of the assimilate stream in ureide-forming legumes (Peoples et
al., 1985).
THE DE NOVO PURINE PATHWAY
Apart from the need for uninfected cells in the
nodule central zone so that uricase function is secure,
the most significant nodule-specific feature of ureide
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Purine Biosynthesis
synthesis is enhanced expression of genes encoding
the enzymes in the purine pathway. Recent studies
have concentrated on the molecular aspects of gene
expression and regulation in relation to N metabolism.
Most of the initial work on the enzymology of the
purine pathway in plants was based on information
gained from other organisms. The pathway, first
characterized in prokaryotes and avian liver, involves 10 enzymatic steps (Fig. 1) to synthesize IMP
(the first product with a complete purine ring) from
PRPP (Zalkin and Dixon, 1992). AMP and GMP are
subsequently formed from IMP. Further research
showed that this model holds in all other organisms,
but the organization of the enzymes differs among
them. In E. coli, nine genes encode the 10 enzyme
activities. These genes are termed the pur genes. (The
nomenclature for the pur genes has changed over
time. In some cases, the genes have been called by the
enzyme name and in others they are termed pur
genes. The bacterial genes are denoted by letters, but
for ease of understanding, we use a numerical nomenclature for plants, e.g. pur1 encodes the enzyme
catalyzing the first step of the pathway. To distinguish genes from different plants the initials of species name are used as a prefix to the gene name, e.g.
the pur1 gene from cowpea is designated Vupur1.) In
E. coli, eight are monofunctional polypeptides, and
one bifunctional polypeptide catalyzes the 10 enzymic reactions (Fig. 3). The pathway organization in
plants is the same, and in this respect, plants are
more similar to prokaryotes than to other eukaryotes
(Fig. 3), in which all but three steps are catalyzed by
either bifunctional or trifunctional enzymes (Zalkin
and Dixon, 1992). The functional domains of these
multifunctional proteins are similar to those of their
monfunctional counterparts in bacteria and plants,
suggesting that during evolution of the eukaryotic
animal cell prokaryotic pur genes were condensed.
WE NEED TO KNOW MORE ABOUT
REGULATION OF THE PURINE PATHWAY
Most work on regulation of purine biosynthesis in
plants has focused on its role in N assimilation. Because expression of some of the pur genes is significantly increased in nodules after N fixation begins
(Smith et al., 1998), a question that has received substantial attention is whether the products of N assimilation are involved in regulating the pathway. The
connection between products of N fixation and activity of the purine pathway was first noted in studies
where nodulated root systems were transiently exposed to an atmosphere of 80% (v/v) Ar:20% (v/v)
O2 (Atkins et al., 1984). Ar replaces N2 in air and,
although the nodules remain physiologically normal,
they do not fix N2 and have no product of fixation to
assimilate. After 3 d of exposure to Ar:O2, activity of
the purine biosynthesis pathway (measured as the
ability of a nodule extract to synthesize IMP when
provided with pathway substrates) was reduced to a
Figure 3. Schematic diagram showing the structural organization of enzymes of the de novo purine biosynthesis pathway
from a range of organisms. In E. coli, each enzyme is monofunctional, except aminoimidazolecarboximide ribonucleotide
transformylase/inosine monophosphate cyclohydrolase, which is bifunctional. AIRC has two subunits encoded by the purE
and purK genes in E. coli (Zalkin and Dixon, 1992). The organization in plants is similar to that in E. coli except that each
enzyme has an N-terminal organelle targeting sequence and the two domains of AIRC are encoded by a single gene (pur6;
Senecoff and Meagher, 1993; Chapman et al., 1994; Arabidopsis Genome Inititiave, 2000). In yeast (Saccharomyces
cerevisiae), AIRC has a similar structure to that in plants (but no targeting sequence) and GARS (step 2) and AIRS (step 5) form
a bifunctional polypeptide encoded by a single gene. All other enzymes are monofunctional. In both Drosophila melanogaster and the vertebrates, a trifunctional polypeptide (with activities for GARS, GART, and AIRS) encoded by a single gene
catalyzes steps 2, 3, and 5. In Drosophila melanogaster, this trifunctional polypeptide has a duplication of the AIRS domain.
A monofunctional GARS protein that is derived by alternative splicing of the GARS/AIRS/GART gene is also expressed in
insects and vertebrates. In these organisms, AIRC and succinoaminoimidazolecarboximide ribonucleotide synthetase (steps
6 and 7) form a bifunctional polypeptide that includes only the purE-like domain of E. coli (Zalkin and Dixon, 1992).
Plant Physiol. Vol. 128, 2002
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
797
Smith and Atkins
fraction of that in control plants. Analysis of intermediates of the pathway during assay suggested a
block after the synthesis of formylglycinamide ribonucleotide (step 3, Fig. 1), but because assays were
not available for the component enzymes and none of
the pur genes had been cloned at that time, the mechanism of regulation could not be determined. A later
study showed activity of AIR synthetase was significantly reduced after 3 h of exposure to Ar:O2 and
was likely to result from a reduction in transcription
of the pur5 gene. These results suggest that pur5 may
be transcriptionally regulated by products of N2 fixation (Atkins and Smith, 2000). Other data supporting a role for products of N assimilation in regulation
of pur gene expression come from experiments where
N solutes were applied to root systems of soybean.
Expression of pur1 was increased 7-fold when Gln
(Kim et al., 1995) was added to the culture medium,
but pur2 and pur3 expression was unaffected
(Schnorr et al., 1996). Treatment with NH4NO3 did
not affect expression of the genes. This result for pur1
in roots conflicts with those from early Ar:O2 experiments where the pathway appeared to be blocked
after step 3 of the pathway (i.e. after the step catalyzed by PRAT, which is encoded by pur1) suggesting that PRAT activity was unaffected by the block in
N fixation. What these results do tell us, however, is
that there is not coordinated regulation of the pathway genes, although the products of N assimilation
are likely to regulate part of the pathway.
There is a noteworthy link between N2 fixation and
the assimilation of fixed N. When purine biosynthesis is blocked by allopurinol (an inhibitor of xanthine
dehydrogenase), fixed N is not assimilated via alternative pathways, such as those that form Asn. N2
fixation is inhibited and the nodules begin to senesce
after 24 h (Atkins et al., 1988). Similarly, where ureide
synthesis was blocked by antisense expression of
uricase (activity reduced by 80%), the transgenic
plants showed symptoms of N deficiency (Lee et al.,
1993). These results indicate that N2 fixation is inhibited under conditions where ureide synthesis is limited, but the nature of the connection is not clear.
IS ARABIDOPSIS HELPFUL?
It is only recently, with the sequencing of the Arabidopsis genome (Arabidopsis Genome Initiative,
2000), that sequences for all genes encoding the purine biosynthesis enzymes became available and, as a
result, that the structure of the plant enzymes could
be deduced. Upstream sequences of the genes are
also available and this will speed research on regulation of the pathway for its “housekeeping roles.”
Each enzyme in Arabidopsis, except PRAT ( pur1), is
encoded by a single gene. Atpur1,1 (AtATase1) and
Atpur1,2 (AtATase2) are differentially expressed (Ito
et al., 1994; although both are expressed in flower
buds, the tissue from which the cDNAs were iso798
lated) with Atpur1,1 expressed in roots and flowers
and Atpur1,2 expressed in flowers, strongly in leaves
but not in roots. To date, there is no evidence that
Atpur1,3 is expressed, but because cDNAs have only
been isolated from flowers, this is not surprising. The
presence of more than one copy of pur1 in Arabidopsis suggests that regulation of the pathway in different tissues may be affected through regulation of
transcription of pur1, and as a consequence the enzyme it encodes, PRAT.
Array studies with Arabidopsis also provide a
valuable source of data that can be analyzed to give
insights into regulation of the pathway. In a recent
report (Girke et al., 2000), expression of pur5 (encoding AIR synthetase) was approximately 19-fold
higher in seeds than in roots or leaves, whereas expression of other pur genes present in the arrays (e.g.
pur1 and pur9) was either reduced or the same. It is
not yet possible to assess the significance of this
observation, but perhaps the pathway is regulated at
this step through transcriptional control of pur5. Assays of the pathway activity in seeds would be necessary to validate this idea but it is consistent with
the transcriptional regulation of pur5 in cowpea nodules exposed to Ar:O2 (Atkins and Smith, 2000).
Analysis of the upstream regions of the pur genes
in Arabidopsis using the PLACE database (http://
www.dna.affrc.go.jp/htdocs/PLACE/; Higo et al.,
1999) showed that most contained a motif, which in
tomato (Lycopersicon esculentum) controls circadian
regulation of the Lhc gene (Piechulla et al., 1998).
However, in an array study by Schaffer et al. (2001),
five of the pur genes (pur1,1; pur4; pur5; pur6; and
pur9) did not show a pattern of expression consistent
with circadian regulation.
Work done with Arabidopsis pur7, in which the
gene promoter was fused to GUS and expression
studied throughout development, showed that expression was highest in actively dividing meristematic tissues, as might be expected (Senecoff et al.,
1996). Auxin treatment also increased expression
from the pur7 promoter, but whether this was a direct
result of auxin on gene expression or an indirect
result of the stimulation of cell division/elongation
by auxin was not addressed (Senecoff et al., 1996).
However, analysis of upstream regions of pur genes
using PLACE identified an auxin-responsive element
(Baumann et al., 1999) in the promoter of pur7 and
also in the promoters of many of the other pur genes
(pur1,2; pur1,3; pur2; pur4; pur8; and pur9). A second
auxin responsive element (Xu et al., 1997) was identified in the upstream regions of pur1,3 and pur4.
The significance of these elements in relation to
plant growth and development is not known and
functional analysis of the pur gene promoters will be
required to validate any predictions derived from
sequence analysis of upstream regions. However,
with information provided by the genome sequence,
the ease of transformation of Arabidopsis and other
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Purine Biosynthesis
tools available for this model species, the analysis
should not be too difficult.
does dual localization occur in all plants and all plant
tissues, or is the localization to both organelles a
strategy to increase purine biosynthesis in nodules
where the flux of purines is much greater than in
other tissues? Because of the low rates of purine
biosynthesis in tissues other than nodules, this question has proved difficult to answer. Plastid transit
peptides and mitochondrial presequences are generally found at the N terminus of a protein (Fig. 3) and
are cleaved to produce a mature protein once the final
location is reached. The targeting sequences that direct
proteins to plastids and mitochondria proteins share
some features in that they have a similar amino acid
composition, but are generally different in their secondary structure. Table I shows an analysis of the
putative targeting sequence of all plant purine enzymes for which genes have been cloned using targeting prediction software, TargetP (Emanuelsson et al.,
2000), and Predotar (http:/www.inra.fr/Internet/
Produits/Predotar/). All but two are predicted to be
targeted to plastids (and in both cases TargetP and
Predotar differ in their predictions). This might be
considered as evidence that dual localization occurs
only in nodules. However, none of the targeting se-
THE PURINE PATHWAY HAS AN UNUSUAL
LOCATION IN NODULES
The localization of the purine biosynthesis pathway in plants is different to that of all other organisms in that it is organelle based (Fig. 2). Synthesis of
purines in animals and bacteria appears to be cytosolic (Gooljarsingh et al., 2001). Early studies (for
review, see Atkins and Smith, 2000), where organelles from either cowpea or soybean nodules
were fractionated, showed that the pathway in plants
was localized to plastids. However, a later study
(Atkins et al., 1997) using more effective fractionation
methods on both Suc and Percoll density gradients,
found that both plastids and mitochondria from cowpea nodules were capable of IMP synthesis from R5P
or PRPP. In addition, activities of a number of pathway enzymes were detected in both organelles.
Localization of the purine biosynthesis enzymes in
two organelles within the same cell raises a number
of questions about their intracellular targeting. First,
Table I. Characteristics of N-terminal extensions of plant purine biosynthetic enzymes
The deduced amino acid sequences of de novo purine biosynthetic enzymes from different plant species were aligned with those of E. coli
using the “Pileup” program of Genetics Computer Group (Madison, WI). Only data from full-length clones were used. The length of the
N-terminal extensions of the plant sequences when compared with the E. coli sequence was calculated (E). Sequences were also analyzed with
the TargetP (version 1.01, Emanuelsson et al., 2000) and Predotar (version 0.5, http:/www.inra.fr/Internet/Produits/Predotar/) software to
determine their predicted subcellular localisations and targeting sequence lengths. Multiple sequences from the same species are distinguished
by a numerical suffix. AICART refers to the bifunctional enzyme AICART-IMPCH. P, Precursor; E, extension; L, subcellular localization predicted
by TargetP; RC, reliability class (1 most reliable, 5 least reliable); TP, targeting peptide length predicted by TargetP; At, Arabidopsis; Gm, soybean;
Nt, Nicotiana tabacum; Vu, cowpea; P, plastid; M, mitochondrion; O, other.
Precursor
AtPRAT1
(AtATase1)
AtPRAT2
(AtATase1)
AtPRAT3
GmPRAT
VuPRAT
AtGARS
VuGARS
AtGART
GmGART1
GmGART2
VuGART
AtFGAR-AT
AtAIRS
VuAIRS
AtAIRC
AtSAICARS
AtASAL
AtAICART
NtAICART
a
Genpept
Accession No.
Gene Name
AAD26498
Atpur1,1
CAA18853
CAB80551
AAA73943
AAC24007
AAB60737
(CAA52778)
AAA74447
CAA52779
CAA65608
–
AAA75367
AAF31730
CAB82696
(AAC37341)
AAC14578
AAC23639
AAA16231
CAB28846
AAC12842
AAG40879
P
TargetP
Predotar
Reference
E
L
RC
TP
532
75
P
1
58
P
Ito et al. (1994)
Atpur1,2
561
34
P
1
53
P
Ito et al. (1994)
Atpur1,3
Gmpur1
Vupur1
Atpur2
566
569
567
532
85
79
77
94
P
P
P
P
1
1
1
1
59
58
58
74
P
P
P
O
Arabidopsis Genome Initiative (2000)
Kim et al. (1995)
–a
Schnorr et al. (1994)
Atpur2
Atpur3
Gmpur3,1
Gmpur3,2
Vupur3
Atpur3
Atpur5
502
292
295
312
312
1387
389
88
77
85
101
98
64
60
P
P
P
P
P
M
P
1
1
2
2
2
3
3
69
65
62
78
73
34
48
O
P
M
P
P
O
P
–
Schnorr et al. (1996)
Schnorr et al. (1996)
Schnorr et al. (1996)
–
Arabidopsis Genome Initiative (2000)
Senecoff and Meagher (1993)b
Vupur5
Atpur6
Atpur7
Atpur8
Atpur9/10
Ntpur9/10
388
645
411
467
545
612
60
89
102
66
13
82
P
P
P
P
P
P
1
3
5
2
5
4
11
68
53
57
26
68
P
P
P
P
O
O
Smith et al. (1998)
Arabidopsis Genome Initiative (2000)
Senecoff et al. (1996)
Arabidopsis Genome Initiative (2000)
Arabidopsis Genome Initiative (2000)
–
No published data, Genbank accession only.
Plant Physiol. Vol. 128, 2002
No. of
Residues
b
Sequence corrected as in CAB82696.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
799
Smith and Atkins
quences for the enzymes from cowpea predict mitochondrial localization and yet the enzymes are present
in mitochondria. Given that genes for eight purine
enzymes from Phaseoloid legumes have been cloned,
it seems unlikely that none of the genes encoding
unique mitochondrial isoforms has been identified.
This suggests that targeting sequences for purine enzymes in ureide-forming legumes may have unique
features, or that import into mitochondria in nodule
cells may be different to that in other tissues.
Second, are there two sets of enzymes, one targeted
to plastids and the second to mitochondria (as is the
case for many proteins localized to different subcellular locations), and if so, are these enzymes products
of the same or different genes? There is little evidence
for multiple copies of the pur genes (pur2, Schnorr et
al., 1996; pur5, Smith et al., 1998). To date, Atpur1 and
Gmpur3 are the only cases for which multiple genes
have been described. Alternative splicing or translation from different start codons can result in two
different proteins being produced as the result of
transcription from a single gene (for review, see Danpure, 1995). In addition, a number of proteins have
recently been identified that are directed to plastids
and mitochondria by the same targeting sequence.
These include glutathione reductase from pea (Pisum
sativum; Creissen et al., 1995), and ferrochelatase-I
(Chow et al., 1997) and methionyl-tRNA synthetase
(Menand et al., 1998) from Arabidopsis.
The best studied of the purine enzymes in terms of
localization is AIR synthetase from cowpea. All the
AIR synthetase cDNAs isolated to date have the same
sequence, and Southern analysis indicates there is
only a single copy of the gene (Smith et al., 1998). The
putative targeting sequence is predicted to target the
protein to plastids (Table I) but is not typical of a
plastid transit peptide. The plastid and mitochondrial isozymes have been purified from cowpea nodules. Although the proteins from the two organelles
differ in the point where they are processed to produce the mature protein, the actual NH2-terminal
amino acid sequences are the same (Goggin, 2001)
and could be derived from the cDNA isolated by
Smith et al. (1998). However, in vitro import assays
with the protein derived from the cDNA indicated
import into chloroplasts but not into mitochondria
(Goggin, 2001).
In the case of the third enzyme of the pathway,
glycinamide ribonucleotide transformylase, two
cDNAs encoding the enzyme have been isolated
from soybean nodules (Schnorr et al., 1996). These
encode proteins that differ only in their N-terminal
amino acid sequence. It is tempting to speculate that
one would be targeted to plastids and the other
to mitochondria. Both are predicted to be localized
to plastids by TargetP but the shorter protein
(GmGART2, Table I) is predicted as mitochondrial by
Predotar. In cowpea, three cDNAs encoding the same
protein have been described (GenBank accession no.
800
AAA75367), each has two in-frame ATGs and it is
possible that translation is initiated at both. The
longer protein, which shares an almost identical targeting sequence with the longer soybean protein, is
predicted to form an amphipathic ␣-helix at its N
terminus, a structure characteristic of mitochondrialtargeted proteins. However, Target P and Predotar
predict plastid localization. The shorter protein is
predicted to be targeted to mitochondria by Target P
but to be targeted to neither organelle by Predotar. In
in vitro import experiments with chloroplasts and
mitochondria, neither of these proteins were imported (Goggin, 2001). There is obviously a need for
more research to elucidate how the purine enzymes
are targeted to mitochondria.
CONCLUSION
Despite the central and indispensable roles for purine biosynthesis in all cells, there is a lack of understanding of the regulation of expression of pur genes
or for localization of pathway enzymes in plants.
Why purine biosynthesis and ureide translocation
occurs as the means to assimilate fixed-N in a small
group of legumes remains a mystery. It is clear that
the host plant does not require this metabolic solution for the assimilation of NH3 or for translocation
of N as ureides, the amides serving the purpose
equally well when the source of N is the soil. Perhaps
the key lies in an interaction in the specific association that develops between this group of legumes
and their symbiotic rhizobia partners. The fact that
purine synthesis is also involved in generating ureides for N storage in some species suggests that
perhaps a more extensive investigation of pathways
for N assimilation in a wider range of plants will
reveal this complex mechanism to be more common
than is presently appreciated.
Received October 10, 2001; accepted December 7, 2001.
LITERATURE CITED
Arabidopsis Genome Inititiave (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815
Atkins CA (1991) Ammonia assimilation and export of
nitrogen from the legume nodule. In MJ Dilworth, AR
Glenn, eds, Biology and Biochemistry of Nitrogen Fixation. Elsevier, Amsterdam, pp 293–319
Atkins CA, Pate JS, Shelp BJ (1984) Effects of short-term
N2 deficiency on N metabolism in legume nodules. Plant
Physiol 76: 705–710
Atkins CA, Sanford PJ, Storer PJ, Pate JS (1988) Inhibition
of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol. Plant Physiol 88:
1229–1234
Atkins CA, Smith PMC (2000) Ureide synthesis in legume
nodules. In EJ Triplett, ed, Prokaryotic Nitrogen Fixation:
A Model System for the Analysis of a Biological Process.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Purine Biosynthesis
Horizon Scientific Press, Wymondham, Norfolk, UK, pp
559–587
Atkins CA, Smith PMC, Storer PJ (1997) Reexamination of
the intracellular localization of de novo purine synthesis
in cowpea nodules. Plant Physiol 113: 127–135
Atkins CA, Storer PJ, Young EB (1991) Translocation of
nitrogen and expression of nodule-specific uricase
(nodulin-35) in Robinia pseudoacacia. Physiol Plant 83:
483–491
Baumann K, De Paolis A, Costantino P, Gualberti G
(1999) The DNA binding site of the Dof protein NtBBF1
is essential for tissue-specific and auxin-regulated expression of the rolB oncogene in plants. Plant Cell 11:
323–333
Brown SM, Walsh KB (1994) Anatomy of the legume
nodule cortex with respect to nodule permeability. Aust
J Plant Physiol 21: 49–68
Chapman KA, Delauney AJ, Kim JH, Verma DPS (1994)
Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia.
Plant Mol Biol 24: 389–395
Cheng XG, Nomura M, Takane K, Kouchi H, Tajima S
(2000) Expression of two uricase (Nodulin-35) genes in a
non-ureide type legume, Medicago sativa. Plant Cell
Physiol 41: 104–109
Chow KS, Singh DP, Roper JM, Smith AG (1997) A single
precursor protein for ferrochelatase-I from Arabidopsis is
imported in vitro to both chloroplasts and mitochondria.
J Biol Chem 272: 27565–27571
Corby HDL (1988) Types of rhizobial nodule and their
distribution among the Leguminoseae. Kirkia 13: 53–123
Coruzzi GM, Zhou L (2001). Carbon and nitrogen sensing
and signaling in plants: emerging matrix effects. Curr
Opinion Plant Biol 4: 247–253
Creissen G, Reynolds H, Xue Y, Mullineaux P (1995)
Simultaneous targeting of pea glutathione reductase and
of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco. Plant J 8: 167–175
Danpure CJ (1995) How can the products of a single gene
be localized to more than one intracellular compartment?
Biochem Biophys Res Comm 203: 866–873
Doyle JJ, Chappill JA, Bailey CD, Kajita T (2000) Towards
a comprehensive phylogeny of legumes: evidence from
RBCL sequences and non-molecular data. Adv Legume
Syst 9: 1–20
Emanuelsson O, Nielsen H, Brunak S, von Heijne G
(2000) Predicting subcellular localization of proteins
based on their N-terminal amino acid sequence. J Mol
Biol 300: 1005–1016
Fujihara S, Yamaguchi M (1978) Effects of allopurinol
[4-hydroxyprazolo(3, 4-d)pyrimidine] on the metabolism of allantoin in soybean plants. Plant Physiol 62:
134–138
Girke T, Todd J, Ruuska S, White J, Benning C, Ohlrogge
J (2000) Microarray analysis of developing Arabidopsis
seeds. Plant Physiol 124: 1570–1581
Goggin DE (2001) Studies on the Localization and Physical
Characteristics of Some Enzymes of de Novo Purine
Plant Physiol. Vol. 128, 2002
Biosynthesis in the Root Nodules of the Tropical Legume
Vigna unguiculata L. (Walp). PhD thesis. The University
of Western Australia
Gooljarsingh LT, Ramcharan J, Gilroy S, Benkovic SJ
(2001) Localization of GAR transformylase in Escherichia
coli and mammalian cells. Proc Natl Acad Sci USA 98:
6565–6570
Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant
cis-acting regulatory DNA elements (PLACE) database.
Nucleic Acids Res 27: 297–300
Ito T, Shiraishi H, Okada K, Shimura Y (1994) Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs. Plant Mol Biol 26:
529–533
Kim JH, Delauney AJ, Verma DPS (1995) Control of de
novo purine biosynthesis genes in ureide-producing legumes: induction of glutamine phosphoribosylpyrophosphate amidotransferase gene and characterization
of its cDNA from soybean and Vigna. Plant J 7: 77–86
Kuzma MM, Hunt S, Layzell DB (1993) Role of oxygen in
the limitation and inhibition of nitrogenase activity and
respiration rate in individual soybean nodules. Plant
Physiol 101: 161–169
Layzell DB, Rainbird RM, Atkins CA, Pate JS (1979)
Economy of photosynthate use in nitrogen-fixing legume
nodules. Plant Physiol 64: 888–891
Lee N-G, Stein B, Suzuki H, Verma DPS (1993) Expression
of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and
affects nitrogen availability to the plant. Plant J 3:
599–606
Menand B, Maréchal-Drouard L, Sakamoto W, Dietrich
A, Winz H (1998) A single gene of chloroplast origin
codes for mitochondrial and chloroplastic methionyltRNA synthetase in Arabidopsis thaliana. Proc Natl Acad
Sci USA 95: 11014–11019
Peoples MB, Pate JS, Atkins CA (1985) The effect of nitrogen source on transport and metabolism of nitrogen in
fruiting plants of cowpea (Vigna unguiculata (L.) Walp.).
J Exp Bot 36: 567–582
Piechulla B, Merforth N, Rudolph B (1998) Identification
of tomato Lhc promoter regions necessary for circadian
expression. Plant Mol Biol 38: 655–662
Rainbird RM, Atkins CA (1981) Purification and some
properties of urate oxidase from nitrogen-fixing nodules
of cowpea. Biochim Biophys Acta 659: 132–140
Reinbothe H, Mothes K (1962) Urea, ureides, and guanidines in plants. Ann Rev Plant Physiol 13: 129–151
Schaffer R, Landgraf J, Accerbi M, Simon VV, Larson M,
Wisman (2001) Microarray analysis of diurnal and
circadian-regulated genes in Arabidopsis. Plant Cell 13:
113–123
Schnorr KM, Laloue M, Hirel B (1996) Isolation of cDNAs
encoding two purine biosynthetic enzymes of soybeanand expression of the corresponding transcipts in
the roots and root nodules. Plant Mol Biol 32: 751–757
Schnorr KM, Nygaard P, Laloue M (1994) Molecular characterization of Arabidopsis thaliana cDNAs encoding three
purine biosynthetic enzymes. Plant J 6: 113–121
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
801
Smith and Atkins
Senecoff JF, McKinney EC, Meagher RB (1996) De Novo
Purine Synthesis in Arabidopsis thaliana: II. The PUR7 gene
encoding 5⬘ phosphoribosyl-4-(N-succinocarboxamide)-5aminoimidazole synthetase is expressed in rapidly dividing tissues. Plant Physiol 112: 905–917
Senecoff J, Meagher RB (1993) Isolating the Arabidopsis
thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants: I. 5⬘-Phosphoribosyl5-aminoimidazole synthetase. Plant Physiol 102: 387–399
Smith PMC, Mann AJ, Goggin DE, Atkins CA (1998) AIR
synthetase in Cowpea Nodules: a single gene product
targeted to two organelles? Plant Mol Biol 36: 811–820
802
Tajima S, Takane K, Nomura M, Kouchi H (2000) Symbiotic nitrogen fixation at the late stage of nodule formation in Lotus japonicus and other legume plants. J Plant
Res 113: 467–473
Thumfort PP, Layzell DB, Atkins CA (1999) Diffusion and
reaction of oxygen in the central tissue of ureide-producing
legume nodules. Plant Cell Environ 22: 1351–1363
Xu N, Hagen G, Guilfoyle T (1997) Multiple auxin response modules in the soybean SAUR 15A promoter.
Plant Sci 126: 193–201
Zalkin H, Dixon JE (1992) De novo purine nucleotide
biosynthesis. Prog Nucleic Acids Res 42: 259–287
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002