J. Plant Physiol. 160.

1271 – 1295 (2003)

 Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp

Review

Purine and pyrimidine nucleotide metabolism in higher plants

Claudio Stasolla1, Riko Katahira2, 3, Trevor A. Thorpe4, Hiroshi Ashihara2, 5 *

1
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada
2
Department of Advanced Bioscience, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, 112-8610, Japan
3
Laboratory of Microbiology, Faculty of Home Economics, Tokyo Kasei Gakuin University, Tokyo, 194-0292, Japan
4
Plant Physiology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada
5
Metabolic Biology Group, Department of Biology, Ochanomizu University, Tokyo, 112-8610, Japan

Received March 27, 2003 · Accepted June 2, 2003

Summary
Purine and pyrimidine nucleotides participate in many biochemical processes in plants. They are
building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary
products, such as sucrose, polysaccharides, phospholipids, as well as secondary products. There-
fore, biosynthesis and metabolism of nucleotides are of fundamental importance in the growth and
development of plants. Nucleotides are synthesized both from amino acids and other small mole-
cules via de novo pathways, and from preformed nucleobases and nucleosides by salvage path-
ways. In this article the biosynthesis, interconversion and degradation of purine and pyrimidine
nucleotides in higher plants are reviewed. This description is followed by an examination of physio-
logical aspects of nucleotide metabolism in various areas of growth and organized development in
plants, including embryo maturation and germination, in vitro organogenesis, storage organ develop-
ment and sprouting, leaf senescence, and cultured plant cells. The effects of environmental factors
on nucleotide metabolism are also described. This review ends with a brief discussion of molecular
studies on nucleotide synthesis and metabolism.

Key words: biosynthesis – catabolism – cultured cells – metabolism – nucleotide – physiological and
molecular studies – plant development – purine – pyrimidine

Abbreviations: APRT = adenine phosphoribosyltransferase. – AK = adenosine kinase. – ARN = ade-

nosine nucleosidase. – dCK = deoxycytidine kinase. – HPRT = hypoxanthine phosphoribosyltransfe-
rase. – NSF = non-shoot forming. – OPRT = orotate phosphoribosyltransferase. – PRPP = 5-phospho-
ribosyl–1-pyrophosphate. – SAH = S-adenosyl-L-homocysteine. – SAM = S-adenosyl-L-methionine. –
SF = shoot forming. – UPRT = uracil phosphoribosyltransferase. – UDPG = UDP-glucose. – UK = uri-
dine kinase. – TK = thymidine kinase

* E-mail corresponding author: ashihara@cc.ocha.ac.jp

0176-1617/03/160/11-1271 $ 15.00/0
1272 Claudio Stasolla et al.

1. Introduction lism during growth and organized development in plants. Re-

Nucleotides are among the most important nitrogen com-
pounds in all living organisms. Although purine and pyrimi-
dine nucleotides are essential precursors for nucleic acids,
as well as metabolites participating in bio-energetic proces-
ses and in the synthesis of macromolecules, including poly-
saccharides, phospholipids, and glycolipids (see Ross 1981),
fewer studies have been performed in higher plants com-
pared with microorganisms (Neuhard and Nygaard 1987) and
animals (Henderson and Paterson 1973). Some reviews on
plant nucleotide metabolism have been published (Ross
1981, Wasternack 1982, Wagner and Backer 1992, Moffatt
and Ashihara 2002), but they deal mainly with the metabolic
pathway itself, or as a part of related topics, such as nucleic
acid metabolism (Sugiura and Takeda 2000) or ureide (Schu-
bert and Boland 1990), and caffeine metabolism (Suzuki and
Waller 1977, Suzuki et al. 1992, Ashihara and Crozier 1999). In
the present review, we have briefly outlined the current status
of purine and pyrimidine studies, and then concentrated on
physiological and biochemical aspects of nucleotide metabo-
cent molecular studies on nucleotide metabolism are also
discussed.
2. Outline of nucleotide metabolism in plants
2.1 Occurrence of nucleobases, nucleosides and
nucleotides
Nucleotide profiles of various plant materials have been de-
termined using high-performance liquid chromatography (see
Wagner and Backer 1992, Ashihara and Crozier 1999). The
adenine nucleotide pool is always the largest, and the sum of
ATP, ADP and AMP is normally 80 – 200 nmol g –1 fresh weight.
The second largest pool is uracil nucleotides. The guanine
nucleotide pool size is usually 10 – 25 % of the adenine nu-
cleotide one, and the cytosine nucleotide pool is the smallest.
In actively growing plant cells adenylate energy charge
([ATP] +1/2 [ADP])/([ATP] + [ADP] + [AMP]), proposed by Atkin-
son (1977), is normally maintained at almost 0.8 (Pradet and
Reymond 1983). Pool size of ATP (160 nmol g –1 fresh weight)

Figure 1. De novo biosynthetic pathway of purine nucleotides in plants. Metabolites: PRA, 5-phosphoribosyl amine; GAR, glycineamide ribonu-
cleotide; FGAR, formylglycineamide ribonucleotide; FGRAM, formylglycine amidine ribonucleotide; AIR, 5-aminoimidazole ribonucleotide; CAIR,
5-aminoimidazole 4-carboxylate ribonucleotide; SCAIR, 5-aminoimidazole-4-N-succinocarboxyamide ribonucleotide; AICAR, 5-aminoimidazole-
4-carboxyamide ribonucleotide; FAICAR, 5-formamidoimidazole-4-carboxyamide ribonucleotide; SAMP, adenylosuccinate; XMP, santhosine-5′-
monophosphate. Enzymes shown are: (1) amido phosphoribosyltransferase, (2) GAR synthetase, (3) GAR formyl transferase, (4) FGAM synthet-
ase, (5) AIR synthetase, (6) AIR carboxylase, (7) SAICAR synthetase, (8) adenylosuccinate lyase, (9) AICAR formyl transferase, (10) IMP cyclo-
hydrolase, (11) SAMP synthetase, (12) IMP dehydrogenase, (13) GMP synthetase.

in typical plant cells, such as Catharanthus roseus supplied
with adequate respiratory substrates, is smaller than the glu-
cose-6-phosphate pool (340 nmol g –1) and larger than fructo-
se-6-phosphate pool (60 nmol g –1) (Kubota and Ashihara
1990).
Cellular pools of nucleosides and free nucleobases are
usually small. For example, in Catharanthus cells, the pool of
adenosine varied from 2 to 9 nmol g –1 fresh weight during cul-
ture, but no free adenine was detected (Yabuki and Ashihara
1991). In contrast, high levels of nucleosides and nucleoba-
ses have been reported in cereal leaves (Sawert et al. 1987,
1988). Uridine, adenosine, guanosine and adenine levels in
barley leaves were 56, 69, 27 and 43 nmol g –1 fresh weight, re-
spectively. These levels were higher than the ATP level
(27 nmol g –1) in the same leaves. Adenylate energy charge in
cereal leaves was between 0.54 and 0.59 (Sawert et al. 1987).
The difference in nucleoside levels between cereals and
other plants may be due to different activities of salvage en-
zymes and/or those of nucleotide catabolism.
2.2 De novo biosynthetic pathway of purine
nucleotides
The de novo biosynthetic pathway of purine nucleotides, AMP
and GMP, from 5-phosphoribosyl-1-pyrophosphate (PRPP) is
shown in Figure 1. Related enzymes from plant sources are
shown in Table 1. Because of space limitation, we have se-
lected references where enzyme properties were best de-
scribed. However, some enzymes described in the table were
demonstrated only in crude extracts. Precise details of this
pathway in plants are still obscure (Sugiura and Takeda
2000), but the same biosynthetic pathway has been estab-
lished in animals and microorganisms (Henderson and Pater-
son 1973, Neuhard and Nyggard 1987, Neuhard and Kelln
1996, Zalkin and Nygaard 1996). In animals, enzymes of the
de novo biosynthetic pathway and those of hydrofolate me-
tabolism are present as a multi-enzyme complex (Chris-
topherson and Szabados 1997). In contrast, such a complex
has not been observed in higher plants. AMP and GMP are
Table 1. Enzymes involved in the de novo biosynthetic pathway of purine nucleotides reported in higher plants.
Enzyme name (step in Fig 1) EC number
Amido phosphoribosyltransferase (1) 2.4.2.14
Adenylosuccinate lyase (8) 4.3.2.2.
AICAR formyl transferase (9) 2.1.2.3
SAMP synthetase (11) 6.3.4.4
IMP dehydrogenase (12) 1.1.1.205
Plant nucleotide metabolism 1273
phosphorylated to nucleoside diphosphates and finally to the
nucleoside triphosphates. Feedback control of the de novo
purine biosynthetic pathway involves at least three steps:
5-phosphoribosylamine synthase activity (step 1 in Fig. 1) is
inhibited by IMP, AMP and GMP; activities of adenylosucci-
nate synthetase (step 11, Fig. 1), and IMP dehydrogenase
(step12, Fig. 1) are inhibited by AMP and GMP, respectively
(see References in Table 1).
2.3 De novo biosynthetic pathway of pyrimidine
nucleotides
The de novo pyrimidine biosynthetic pathway (orotate path-
way) is defined as the formation of UMP from carbamoyl
phosphate. The orotate pathway consists of six reactions as
shown in Figure 2 and Table 2. In mammals and many other
eukaryotes, the first three enzymes, carbamoylphosphate
synthetase, aspartate transcarbamoylase and dihydroorotase
(steps 1– 3, Fig. 2) are present as a multifunctional protein
called the CAD protein, after the initial letters of the three con-
stituent enzymes (Christopherson and Szabados 1997). How-
ever, no such complex has thus far been detected in plants
(see Moffatt and Ashihara 2002). Although there are two dif-
ferent types of carbamoyl phosphate synthetases that pro-
vide substrates for pyrimidine and arginine biosynthesis in
most eukaryotes, including mammals, only one type has thus
far been identified in higher plants (see Wagner and Backer
1992). Therefore, the plant enzyme evidently provides carba-
moyl phosphate for both pathways. UMP is a feedback inhib-
itor of plant carbamoyl phosphate synthetase, but this inhibi-
tion is overcome by ornithine, leading to the utilization of car-
bamoylphosphate for arginine biosynthesis (O’Neal and Nay-
lor 1976). The second enzyme, aspartate transcarbamoylase,
is also inhibited by high concentrations of UMP. This seems to
be a feedback control of pyrimidine biosynthesis. The fourth
enzyme (step 4 in Fig. 2), dihydroorotate dehydrogenase, is
not well characterized. Subcellular localization studies with
hetrotrophically cultured tomato cells suggested that it is lo-
cated in mitochondria (Miersch et al. 1986). The fifth and sixth
Plant source Reference
Glycine max (nodules) Reynolds et al. (1984)
Triticum aestivum (germ) Hatch (1966)
Pisum sativum (seedlings) Iwai et al. (1972)
Zea mays (seedlings) Walters et al. (1997)
Arabidopsis thaliana Prade et al. (2000)
Triticum aestivum Prade et al. (2000)
Pisum sativum (seeds) Turner and King (1961)
Vigna unguiculata (nodules) Atkins et al. (1985)
Camellia sinensis (leaves) Nishimura and Ashihara (1993)
Catharanthus roseus (cells) Nishimura and Ashihara (1993)
1274 Claudio Stasolla et al.

Figure 2. De novo biosynthetic pathway of pyrimidine nucleotides in plants. Enzymes shown are: (1) carbamoyl phosphate synthetase, (2) aspar-
tate transcarbamoylase, (3) dihydroorotase, (4) dihydroorotate dehydrogenase, (5) – (6) UMP synthase (orotate phosphoribosyltransferase plus
orotidine-5′-phosphate decarboxylase), (7) UMP kinase, (8) nucleoside diphosphate kinase, (9) CTP synthetase.

Table 2. Enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides reported in higher plants.

Enzyme (step in Fig 2) EC number Plant source Reference

Carbamoyl phosphate syntehtase (1) 6.3.5.5 Phaseolus aureus Ong and Jackson (1972)
Pisum sativum (shoots) O’neal and Naylor (1976)
Aspartate transcarbamoylase (2) 2.1.3.2 Phaseolus aureus (seedlings) Rao et al. (1979)
Triticum aestivum (germ) Khan et al. (1999)
Dihydroorotate dehydrogenase (4) 1.3.99.11 Lycopersicon esculentum (cells) Miersch et al. (1986)
Orotate phosphoribosyltransferase (5) and 2.4.2.10 Phaseolus mungo (seedlings) Ashihara (1978)
Orotidine-5’-phosphate decarboxylase (6) 4.1.1.23 Lycopersicon esculentum Walther et al. (1984)
(cultured cells)
UMP/CMP kinase (7) 2.7.4.14 Arabidopsis thaliana Zhou et al. (1998)
Nucleoside-diphosphate kinase (8) 2.7.4.6 Spinacia oleracea (leaves) Nomura et al. (1991)
Avena sativa (seedlings) Sommer and Song (1994)

enzymes, orotate phosphoribosyltransferase (step 5, Fig. 2) 2.4 Biosynthesis of deoxyribonucleotides

and orotidine-5′-monophosphate decarboxylase (step 6,
Fig. 2), are present as a single polypeptide; as a result, a new
term, UMP synthase, has been given this protein, which is ob-
served in both plants and animals (Santoso and Thornburg
1998). UMP produced by the orotate pathway is further phos-
phorylated by UMP kinase (step 7, Fig. 2) and nucleoside di-
phosphate kinase (step 8, Fig. 2) to UTP via UDP. CTP is
formed from UTP by a one-step reaction catalysed by CTP
synthetase (step 9, Fig. 2).
The reduction of the ribose moiety of the ribonucleotide di-
phosphates (NDP) is catalysed by a single plant ribonucleo-
tide reductase (step 1, Fig. 3), and deoxyribonucleoside di-
phosphates (dNDPs) are produced (see Wagner and Backer
1992). With the exception of dUDP, dNDPs are further phos-
phorylated and the resultants, dCTP, dATP and dGTP are uti-
lized as direct precursors for DNA synthesis. In plants, dUDP
formed by the ribonucleotide reductase is first converted to
 Plant nucleotide metabolism 1275

Figure 3. De novo biosynthetic pathway of deoxy-

nucleotides in plants. (1) Ribonucleotide reduc-
tase, (2) nucleoside diphosphate kinase, (3)
dUTP pyrophosphatase, (4) thymidylate syn-
thase, (5) nucleoside monophosphate kinase
and/or dTMP kinase.

Table 3. Enzymes involved in the salvage pathways of purine nucleotides reported in higher plants.

Enzyme name (step in Fig 4 a) EC number Plant source Reference

Adenine phosphoribosyltransferase (1) 2.4.2.7 Helianthus tuberosus (shoots)
Catharanthus roseus (cells)
Lycopersicon esculentum
(roots, leaves)
Brassica juncea (leaves)
Arabidopsis thaliana (leaves)
Hevea brasiliensis (latex)
Hypoxanthine-guanine 2.4.2.8 Helianthus tuberosus (shoots)
phosphoribosyltransferase (2)
Adenosine phosphorylase (3) 2.4.2.- Triticum aestivum (germ)
Adenosine kinase (5) 2.7.1.20 Lupinus luteus (seeds)
Prunus persica (flower buds)
Arabidopsis thaliana
Nucleoside phoshpotransferase (6) 2.7.1.77 Daucus carota (roots)
Lupinus luteus (cotyledons)
Hordeum vulgare (seedlings)
Inosine-guanosine kinase (7) 2.7.1.73 Helianthus tuberosus (tubers)
Inosine-guanosine phosphorylase (8) 2.4.2.1 Triticum aestivum (germ)
Le Floc’h and Lafleuriel (1978)
Hirose and Ashihara (1983 a)
Burch and Stuchbury (1986)
Moffatt and Somerville (1990)
Lee and Moffatt (1993)
Gallois et al. (1996)
Le Floc’h and Lafleuriel (1983 b)
Chen and Petschow (1978)
Guranowski (1979 a)
Faye and Le Floc’h (1997)
Mofatt et al. (2000)
Rodgers and Chargaff (1972)
Guranowski (1979 b)
Prasher et al. (1982)
Combés et al. (1989)
Chen and Petschow (1978)

dUTP, and then hydrolysed to dUMP (step 3, Fig. 3) (Pardo
and Gutiérrez 1990).
2.5 Synthesis of thymidine nucleotides
Thymidine nucleotide (dTMP) is synthesized from dUMP by
thymidylate synthase (step 4, Fig. 3). In this reaction, N 5,
N 10-methyltetrahydrofolate produced by dihydrofolate reduc-
tase acts both as donor of the methyl group and reducing
agent. A bifunctional protein that consists of thymidylate syn-
thase and dihydrofolate reductase has been detected in
plants (Lazar et al. 1993). Conversion of dTMP to dTTP oc-
curs by sequential reactions catalysed by nucleoside mono-
phosphate kinase (step 5, Fig. 3) and nucleoside diphos-
phate kinase (step 2, Fig. 3).
2.6 Purine salvage
Utilization of preformed purine bases and nucleosides for nu-
cleotide synthesis occurs through the salvage pathway. Pu-
rine nucleosides and nucleobases arise from the intercellular
breakdown of unstable RNA and nucleotides, and in some
cases, originate from exogenous sources as catabolic prod-
ucts of nucleic acids and nucleotides in decaying cells (see
Wasternack 1982, Bray 1983). Adenosine released from hy-
drolysis of S-adenosyl-L-homocysteine in the S-adenosyl-L-
methionine cycle and adenine released in the methionine cy-
cle of ethylene synthesis are also utilized as a substrate for
purine salvage (see Wasternack 1982, Ashihara and Crozier
1999, Crozier et al. 2000, Moffatt and Ashihara 2002). Some
of the enzymes involved in purine salvage have been found in
higher plants (Table 3).
1276 Claudio Stasolla et al.

Figure 4. Salvage reactions of (A) purine and (B) pyrimidine bases and nucleosides in plants. Enzymes shown are: (A)-(1) adenine phosphoribo-
syltransferase, (2) hypoxanthine-guanine phoshoribosyltransferase, (3) adenosine phosphorylase, (4) adenosine nucleosidase, (5) adenosine
kinase, (6) non-specific nucleoside phosphotransferase, (7) inosine-guanosine kinase, (8) inosine-guanosine phosphorylase, (9) inosine-guano-
sine nucleosidase. (B)-(1) uracil phosphoribosyltransferase, (2) uridine/cytidine kinase, (3) non-specific nucleoside phosphotransferase, (4)
deoxycytidine deaminase, (5) deoxycytidine kinase, (6) thymidine kinase, (1) uridine phosphorylase, (8) uridine nucleosidase.

Three purine bases, adenine, guanine and hypoxanthine,
are salvaged to AMP, GMP and IMP, respectively. Two distinct
enzymes, adenine phosphoribosyltransferase (step 1, Fig.
4 A) and hypoxanthine/guanine phosphoribosyltransferase
(step 2, Fig. 4 A) participate in these salvage reactions. Purine
bases also may be salvaged to nucleotides via nucleosides.
Adenosine phosphorylase (step 3, Fig. 4 A) and inosine-gua-
nosine phosphorylase (step 8, Fig. 4 A) convert adenine,
hypoxanthine and guanine to their respective ribonucleosides
using ribose-1-phosphate. Activities of these enzymes are
very low in higher plants (see Wagner and Backer 1992, Ashi-
hara and Crozier 1999, Moffatt and Ashihara 2002). Thus, the
production of ribonucleotides by these methods may only
play a minor role in purine salvage.
Purine nucleosides, adenosine, guanosine and inosine, are
salvaged by kinases and/or nucleoside phosphotransferase.
Adenosine kinase (step 5, Fig. 4 A) is distributed ubiquitously
and its activity is usually high, whereas, inosine-guanosine ki-
nase (step 7, Fig. 4 A) has been found in only a limited num-
ber of plant species. In some plants, inosine and guanosine
are mainly salvaged by the non-specific nucleoside phospho-
transferase (step 6, Fig. 4 A) (see Wagner and Backer 1992,
Ashihara and Crozier 1999).
2.7 Pyrimidine salvage
Some of the pyrimidine bases and nucleosides produced as
degradation products of nucleotide and nucleic acids are uti-
lized for generation of pyrimidine nucleotides (see Ross 1981,
Wasternack 1982, Wagner and Backer 1992). Uracil, one of
the pyrimidine bases, is salvaged by uracil phosphoribosyl-
transferase (step 1, Fig. 4 B). No salvage activity has yet been
detected for cytosine in plants (Ross 1981, Katahira and Ashi-
hara 2002), although cytosine is converted to uracil by cyto-
sine deaminase and salvaged in some microorganisms (Ipata
et al. 1971, Sakai et al. 1976, Katsuragi et al. 1986). Pyrimidine
nucleosides, uridine, cytidine, deoxycytidine and thymidine,
are salvaged to their respective nucleotides, UMP, CMP,
dCMP and dTMP. A single enzyme, uridine/cytidine kinase
(step 2, Fig. 4 B) that is present in all plants investigated to
date, phosphorylates uridine and cytidine. Deoxycytidine ki-
nase and thymidine kinase (steps 5 and 6 respectively,
 Plant nucleotide metabolism 1277

Table 4. Enzymes involved in the salvage pathways of pyrimidine nucleotides reported in higher plants.

Enzyme (step in Fig 4b) EC number Plant source Reference

Uracil phosphoribosyltransferase (1) 2.4.2.9 Pisum sativum (epicotyls) Bressan et al. (1978)
Uridine/Cytidine kinase (2) 2.7.1.48 Zea mays (seeds) Deng and Ives (1975)
Deoxycytidine deaminase (4) 3.5.4.14 Zea mays (leaves) Le Floc’h and Guillot (1974)

Figure 5. Catabolism of (A) purine nucleotides and (B) pyrimidine nucleotides in plants. Enzymes shown are: (A)-(1) AMP deaminase, (2) IMP de-
hydrogenase, (3) 5′-nucleotidase and/or phosphatase, (4) inosine-guanosine nucleosidase, (5) guanosine deaminase, (6) guanine deaminase,
(7) xanthine dehydrogenase, (8) uricase, (9) allantoinase, (10) allantoicase, (11) ureidoglycolate lyase, (12) urease, (13) allantoin deaminase, (14)
ureidoglycine amidohydrolase, (15) ureidoglycolate hydrolase. (B)-(1) 5′-nucleotidase and/or phosphatase, (2) cytidine deaminase, (3) uridine
nucleosidase, (4) dihydrouracil dehydrogenase, (5) dihydropyriminase, (6) β-ureidopropionase, (7) thymidine phosphorylase and/or thymidine
nucleosidase.

Fig. 4 B) may be present in plants, but details of their proper-
ties have not yet been elucidated. Non-specific nucleoside
phosphotransferase (step 3, Fig. 4 B) activity also participates
in the salvage of pyrimidine nucleosides, as well as purine
nucleosides. Some of the enzymes of pyrimidine salvage
have been reported in higher plants (Table 4).
2.8 Catabolism of purine nucleotides
Plants possess the complete oxidative purine catabolic path-
way to CO2 and NH3 via uric acid and allantoin (Fig. 5 A and
Table 5, see Schubert and Boland 1990, Ashihara and Crozier
1999). A key starting compound of purine catabolism is xant-
hine; thus, all purine nucleotides must be converted to xant-
hine before their purine ring cleavage is initiated. Deamina-
tion, dephosphorylation and glycosidic bond cleavage are
involved in this process. In contrast to animals, adenosine
deaminase has generally not been found in most plants,
although very low adenosine deaminase was detected in
roots and foliage of alfalfa (Medicago sativa) plants (Edwards
1996); thus, AMP deaminase and guanosine deaminase are
the predominant deamination enzymes for adenine and gua-
1278 Claudio Stasolla et al.

Table 5. Enzymes involved in the catabolic pathway of purine nucleotides reported in higher plants.

Enzyme name (step in Fig 3) EC number Plant source Reference

AMP deaminase (1) 3.5.4.6 Spinacia oleracea (leaves) Yoshino and Murakami (1980)
Helianthus tuberosus (tubers) Le Floc’h and Lafleuriel (1983 a)
Catharanthus roseus (cells) Yabuki and Ashihara (1992)
Pisum sativum (seedlings) Dancer et al. (1997)
5’-Nucleotidase (3) 3.1.3.5 Zea mays (seedlings shoot) Carter and Tipton (1985)
Lycopersicon esculentum (roots, leaves) Burch and Stuchbury (1986)
Arachis hypogaea (cotyledons) Gupta and Sharma (1996)
Inosine-guanosine nucleosidase (4) 3.2.2.2 Helianthus tuberosus (shoots) Le Floc’h and Lafleuriel (1981)
Lupinus luteus (seeds) Granowski (1982)
Purine nucleosidase 3.2.2.1 Vigna unguiculata (nodules) Atkins et al. (1989)
Adenosine nucleosidase 3.2.2.7 Lupinus luteus (seeds) Abusamhadneh et al. (2000)
Guanosine deaminase (5) 3.5.4.15 Camellia sinensis (leaves) Negishi et al. (1994)
Guanine deaminase (6) 3.5.4.3 Camellia sinensis (leaves) Negishi et al. (1994)
Xanthine dehydrogenase (7) 1.1.1.204 Glycine max (nodules) Triplett et al. (1982)
Uricase (8) 1.7.3.3 Vigna unguiculata (nodules) Rainbird and Atkins (1981)
Phaseolus vulgaris (nodules) Sánchez et al. (1987)
Glycine max (nodules) Kahan and Tipton (1997)
Allantoinase (9) 3.5.2.5 Glycine max (leaves, fruits) Thomas et al. (1983)
Glycine max (seeds) Webb and Lindell (1993)
Glycine max (nodules, cotyledons) Bell and Webb (1995)
Ureidoglycolate lyase (11) 4.3.2.3 Cicer arietinum (pods) Munoz et al. (2001)
Urease (12) 3.5.1.5 Morus alba (leaves) Hirayama et al. (2000)
Ureidoglycolate hydrolase (15) 3.5.3.19 Glycine max (seedcoats) Winkler et al. (1988)
Phaseolus vulgaris (fruits) Wells and Lees (1991)

Table 6. Enzymes involved in the catabolic pathway of pyrimidine nucleotides reported in higher plants.

Enzyme (step in Fig. 5) EC number Plant source Reference

Cytidine deaminase (2) 3.5.4.5 Arabidopsis thaliana Vincenzetti et al (1999)

Arabidopsis thaliana Faivre-Nitschke et al. (1999)
Uridine nucleosidase (3) 3.2.2.3 Phaseolus radiatus (seedlings) Achar and Vaidyanathan (1967)
Pisum sativum (cotyledons) Murray and Ross (1971)
β-ureidopropionase (6) 3.5.1.6 Zea mays (seedlings) Walsh et al. (2001)

nine nucleotides respectively. Guanine deaminase is also
detected in plants, but its activity is generally low. GMP
reductase, which catalyses the conversion of GMP to IMP, is
not present in plants. There are many enzymes for the
dephosphorylation reaction. Various phosphatases, 3′-
nucleotidase and 5′-nucleotidase appear to produce purine
nucleosides. Adenosine nucleosidase, inosine-guanosine
nucleosidase seem to participate in glycosidic bond cleav-
age.
Xanthine is converted to uric acid by xanthine dehydrogen-
ase (step 7, Fig. 5 A). Uricase catalyses the formation of allan-
toin, and allantoic acid is produced by an allantoinase-cata-
lysed reaction (steps 8 and 9, Fig. 5 A). Some plant organs,
such as roots of the tropical legumes and maple (Acer sp.)
trees, accumulate allantoin and/or allantoic acid, which play
an important role in the storage and translocation of nitrogen
(see Schubert and Boland 1990). Different metabolic fates of
allantoic acid are proposed in plants. In the classic allan-
toicase pathway, allantoic acid is degraded to CO2, NH3 and
glyoxylate via urea and ureidoglycolate (steps 10–12, Fig.
5 A). More recently, an alternative route, the allantoic acid
amidohydrolase pathway, has been proposed in which allan-
toic acid is initially converted to ureidoglycine, CO2 and NH3
(step 13. Fig. 5 A). The NH3 is released directly and urea for-
mation is not involved in the subsequent catabolism to gly-
oxylate (steps 14 and 15, Fig. 5 A) (Winkler et al. 1988).
2.9 Catabolism of pyrimidine nucleotides
In plants, the pyrimidine bases, uracil and thymine, are cata-
bolized by a reductive pathway (see Wasternack 1978),
whereas no catabolic pathway of cytosine has been ob-

served (see Ross 1981). Thus, catabolism of CMP must take
place after conversion of cytidine to uridine (step 2, Fig. 5 B).
Plants have cytidine deaminase, but not cytosine deaminase,
so conversion at the nucleoside level is essential. Uracil and
thymine are catabolized by the same three sequential reac-
tions (steps 4 – 6, Fig. 5 B). The end products of this catabolic
pathway are either β-alanine or β-aminoisobutyrate. In both
cases, CO2 and NH3 are produced as by-products. Only
three enzymes of the pyrimidine catabolic pathway have
been demonstrated in higher plants to date (Table 6).
2.10 Related pathways
Purine nucleotides are also precursors for cytokinin and pu-
rine alkaloid biosynthesis. Therefore, nucleotide metabolism
has unique roles in plants. For reviews on these topics, recent
publications (Kaminek et al. 1992, Ashihara and Crozier 1999,
2001, Mok and Mok 2001) are available.
3. Physiological aspects of nucleotide
metabolism in plants
Relatively few studies deal with the synthesis and metabolism
of purines and pyrimidines in plants. A majority of the investi-
gations on nucleotide metabolism have been carried out by
following the metabolic fate of radio-labeled purine and pyri-
midine bases and nucleosides, which are readily taken up by
the tissue and metabolised to nucleotides or degraded to
CO2 and NH3. Such studies have been of great use in deter-
mining the activity of the de novo, salvage, and degradation
pathways of both purine and pyrimidine metabolism during
different stages of plant growth and development. Today,
major studies on this topic have been conducted during ma-
turation and germination of somatic and zygotic embryos,
shoot organogenesis in vitro, development of storage organs
and sprouting, fruit ripening, and leaf senescence.
3.1 Embryo maturation
Studies on nucleotide metabolism during embryogenesis
have been carried out mainly on (asexual) somatic embryos,
because their zygotic counterparts are embedded in the ma-
ternal tissue, and are difficult to isolate. In our laboratories,
white spruce (Picea glauca) and carrot (Daucus carota) so-
matic embryogenesis in vitro have been extensively utilized
as model systems for this purpose.
Both purine salvage and degradation pathways, as investi-
gated by following the metabolic fate of [8-14C]adenine and
[8-14C]adenosine, substrates of the salvage pathway (see
Fig. 4), and [8-14C]inosine, intermediate of the degradation
pathway (see Fig. 5 A), operate during the development of
white spruce somatic embryos (Ashihara et al. 2001 a). From
this study, it emerged that changes in salvage activity may
Plant nucleotide metabolism 1279
represent a metabolic switch required for the termination of
cell proliferation and the initiation of embryo development. Ex-
tensive utilization of adenine and adenosine for nucleotide
and nucleic acid synthesis, as well as high activities of the re-
spective salvage enzymes, adenine phosphoribosyltransfe-
rase (APRT) and adenosine kinase (AK), was in fact ob-
served during the initial phases of embryo development,
characterized by rapid cell proliferation. During the sub-
sequent stages, characterized by a reduction of cell prolifera-
tion and the initiation of somatic embryo development, de-
creases in salvage activity of both adenine and adenosine
were observed (Ashihara et al. 2001 a). Changes in purine
metabolism also seem to be important during the imposition
of desiccation on the white spruce somatic embryos, required
for the termination of the developmental mode and the initia-
tion of germination (Kermode 1990). During this transition, in
fact, although the incorporation of both supplied adenine and
adenosine declined, the activity of APRT was found to in-
crease (Stasolla et al. 2001 a). Although operative throughout
the maturation period of white spruce somatic embryos, the
degradation pathway, estimated by the rate of inosine degra-
dation, does not appear to be correlated to any particular
stage of development.
Studies on pyrimidine metabolism conducted on maturing
white spruce somatic embryos demonstrated that the de
novo pathway (see Fig. 2) is very active throughout the em-
bryogenic process, as more than 80 % of supplied orotic acid
was utilized for nucleotide and nucleic acid synthesis at the
different stages of embryo development (Ashihara et al.
2001 b). The salvage pathway (see Fig. 4 B) was also oper-
ative, and uridine was a better precursor for nucleotides and
nucleic acids than was uracil. The extensive catabolism of
uracil (see Fig. 5 B), degraded to CO2 via β-ureidopropionate,
was mainly due to the low activity of uracil phosphoribosyl-
transferase (UPRT) in relation to that of uridine kinase (UK)
(Ashihara et al. 2001 b). As the changing pattern of these en-
zymes did not correlate with the utilization of their respective
precursors, it was suggested that other fine control mecha-
nisms, including the fluctuation of levels of substrates and/or
effectors might be a determinant for the control of pyrimidine
metabolism during embryo development (Ashihara et al.
2001 b). As also observed for APRT, the activity of UK was
found to increase during the subsequent imposition of desic-
cation (Stasolla et al. 2001a). High activity of this enzyme was
also observed in white spruce zygotic embryos dissected
from dry seeds (Stasolla et al. 2002). The participation of
these two enzymes for the enlargement of the overall nucleo-
tide pool at germination will be discussed in the next section.
Differences in pyrimidine metabolism seem to exist during
the early stages of maturation between dried seeds of coni-
fers and flowering plants. An active pyrimidine de novo path-
way, as estimated by the utilization of orotic acid for nucleo-
tide and nucleic acid biosynthesis was operative in both so-
matic (Stasolla et al. 2001a) and zygotic (Stasolla et al. 2002)
white spruce embryos, but not in dried black gram (Phaseo-
1280 Claudio Stasolla et al.
lus mungo) seeds (Ashihara 1977). In black gram seeds, this
situation was associated with a low content of 5-phosphoribo-
syl-1-pyrophosphate (PRPP) (Ashihara and Kameyama 1989).
Although not directly measured, PRPP does not appear to be
a limiting factor in dried conifer embryos, as the activity of
PRPP synthase doubled during the imposed drying treatment
in white spruce somatic embryos (Stasolla et al. 2001a).
Despite the paucity of information regarding the biosynthe-
sis of deoxyribonucleotides in plants, the existence of a de
novo and salvage pathway during the embryogenic process
in both angiosperms and gymnosperms has been demon-
strated (Schimpff et al. 1978, Stasolla et al. 2003 a, b). Evi-
dence that the de novo pathway of deoxyribonucleotide bio-
synthesis is present in plant embryos comes from studies of
Schimpff et al. (1978), who measured ribonucleotide reduc-
tase activity in wheat (Triticum aestivum) embryos. Incorpora-
tion of radioactivity from 14C-labeled cytidine into the DNA
fraction of maturing white spruce and carrot somatic embryos
(Stasolla et al. 2003 a, b) also supports this concept. In carrot,
the increased utilization of cytidine for DNA synthesis as ma-
turation progresses, clearly indicates that de novo synthesis
of deoxyribonucleotides may be required in support of active
embryonic growth (Stasolla et al. 2003 b).
Tracer experiments conducted with thymidine and deoxy-
cytidine on white spruce and carrot also indicate that the sal-
vage synthesis of deoxyribonucleotides is operative during
embryo development. Both precursors are in fact rapidly met-
abolised by the tissue and incorporated into nucleotides and
nucleic acids. Compared to deoxycytidine, thymidine was
more actively incorporated into DNA rather than RNA in both
systems (Stasolla et al. 2003 a, b). Salvage synthesis of de-
oxyribonucleotides may be required during the initial stages
of embryo development, as low salvage activity for both de-
oxycytidine and thymidine was observed in habituated carrot
cells that were unable to undergo embryo development upon
removal of exogenous 2,4-D (Stasolla et al. 2003 b).
3.2 Embryo germination
In the past, much work related to nucleic acid synthesis dur-
ing seed germination has been published (see Deltour 1985).
Nevertheless, information concerning purine and pyrimidine
metabolism during germination is relatively limited. Availabil-
ity of nucleotides during the early phases of imbibition seems
to be a critical factor for successful embryo germination. Be-
sides their involvement in the myriad of bio-energetic proces-
ses required for the mobilization of storage products, active
nucleotide biosynthesis is also needed to provide the em-
bryos with sufficient purine and pyrimidine nucleotides to
support nucleic acid synthesis (Deltour 1985). As such, it is
not surprising that a tight regulation between de novo, sal-
vage, and degradation pathways of both purines and pyrimi-
dines exists at germination.
The metabolic pattern of purine metabolism during germi-
nation, as proposed by Ashihara (1983), can be divided in
three distinct phases: (1) «unmetabolized phase», (2) «sal-
vage phase», and (3) «ureide formation phase». During the
early stages of germination, the total uptake of purine precur-
sors, as well as their incorporation into the different cellular
fractions is generally low (Stasolla et al. 2001 b). In black
gram cotyledons, for example, more than 50 % of both ade-
nine and adenosine and 30 % of guanine and hypoxanthine
taken up by the tissue remained unmetabolized (Ashihara
1983). A similar result was also observed in germinating white
spruce embryos fed with adenine, adenosine, and inosine
(Stasolla et al. 2001 b). Such results may be ascribed to the
absence of full tissue hydration during the first hours of imbi-
bition; therefore, the whole machinery involved in the utiliza-
tion of purine precursors is probably not fully functional.
The second phase of purine metabolism at germination is
the «salvage phase». The first hints of salvage activity follow-
ing seed imbibition came from the studies conducted by
Bomsel and Pradet (1968) and Cheung and Marcus (1976),
who observed an increase of the nucleoside triphosphate
pool at the expense of the endogenous AMP and GMP. The
importance of the purine salvage pathway at germination was
further demonstrated by Anderson (1977a), who observed an
increase of ATP and, to a lesser extent, GTP in soybean
(Glycine max) axes incubated in the presence of adenine and
adenosine. Furthermore, increase in PRPP, an important
phosphoribosyl donor for nucleotide synthesis, was observed
in black gram seeds (Ashihara and Kameyama 1989). In-
creasing concentrations of ATP were also found in other sys-
tems, including germinating black gram seeds (Ashihara and
Kameyama 1989), pine (Pinus mugo) pollen (Nygaard 1973),
bacterial spores (Setlow and Kornberg 1970), and soybean
embryos (Anderson 1977 a). A positive correlation between
ATP content, and seed viability and seedling size was ob-
served in many species, including ryegrass (Lolium multiflo-
rum), rape seed (Brassica napus), and crimson clover (Trifo-
lium incarnatum) (Ching 1973). Comparative studies between
control and deteriorated soybean seeds, characterized by a
low percentage of conversion, revealed a higher endogenous
amount of ATP in control seeds (Anderson 1977a). The author
also suggested that the impaired ability to generate adeny-
late nucleotides, rather than loss of specific activities of en-
zymes involved in biosynthetic processes, was responsible
for the reduced germination frequency of deteriorated seeds
(Anderson 1977b).
Production of purine nucleotides via the salvage pathway
(see Fig. 4 A) at germination occurs mainly at the expenses of
adenine, adenosine, guanine, and guanosine (Anderson
1977 a, Guranowski 1979 a, Guranowski and Barankiewicz
1979, Ashihara 1983, Ashihara et al. 1997, Nobusawa and
Ashihara 1983, Stasolla et al. 2001 b). During germination of
white spruce somatic embryos (Stasolla et al. 2001b), as well
as of black gram seeds (Nobusawa and Ashihara 1983),
wheat embryos (Price and Murray 1969), and lupin seeds
(Lupinus luteus) (Guranowski and Barankiewicz 1979), sal-
vage of adenine is mainly catalysed by APRT. The activity of

this enzyme increased two-fold over a 2 d period in wheat
embryos (Price and Murray 1969) and a 4 d period in white
spruce somatic embryos (Stasolla et al. 2001 b). The altern-
ative route of adenine salvage, involving a two step reaction
catalysed by nucleoside phosphorylase and AK, is not likely
to operate at germination, because no nucleoside phos-
phorylase was detected in either dry and germinating lupin
seeds (Guranowski and Barankiewicz 1979).
The two mechanisms of adenosine salvage, the single step
reaction mediated by AK and the two step reaction catalysed
by adenosine nucleosidase (ARN) and APRT (see Fig. 4 A)
are both active at germination, but they operate at different
times. During the initial phases of germination, there are indi-
cations that adenosine salvage is mainly mediated by AK.
The activity of this enzyme, found in extracts of lupin and
black gram seedlings, especially in the cotyledons (Gura-
nowski 1979 b, Guranowski and Barankiewicz 1979, Nobusawa
and Ashihara 1983), increased during germination of white
spruce somatic embryos (Stasolla et al. 2001 b), whereas it
remained constant in germinating wheat embryos (Price and
Murray 1969). The alternative route of adenosine salvage ap-
parently becomes operative only during the later stages of
germination, as no ARN activity was detected in dried white
spruce somatic embryos (Stasolla et al. 2001 a) nor in dried
lupin seeds (Guranowski and Pawelkiewicz 1978). In white
spruce the presence of this enzyme only appeared 4 d after
imbibition (Stasolla et al. 2001 b), whereas in cotyledons of
germinating lupin seeds ARN activity was first detected at
day 2 and it reached its maximum activity around day 4 (Gu-
ranowski and Pawelkiewicz 1978).
Together with adenine and adenosine, there are a few re-
ports indicating that salvage of guanine and guanosine oc-
curs at germination. Ashihara (1983) observed an extensive
utilization of supplied guanine into nucleosides and nucleo-
tide fractions of germinating black gram seed. This was as-
cribed to the activity of GPRT, which was detected in both
embryonic axes and cotyledons (Ashihara 1983). In another
study, Ashihara et al. (1997) also demonstrated a rapid incor-
poration of guanosine into nucleotides and nucleic acids of
2-day-old black gram seedlings. Compared to that of adenine
and adenosine, however, our understanding of the impor-
tance of guanine and guanosine salvage is limited.
Active synthesis of purine nucleotides via the salvage
pathway appears to be critical for the resumption of growth at
germination. In white spruce somatic embryos, treatments
that improve the conversion of the embryos into viable plant-
lets (Stasolla and Yeung 1999), also increase the activity of
the salvage pathway (Stasolla et al. 2001 b). The contribution
of the salvage synthesis to the overall nucleotide pool ap-
pears to be critical during the early phases of germination, as
(1) the de novo pathway of purine nucleotide synthesis, dem-
onstrated to be operative in soybean axes (Anderson 1979)
and wheat embryos (Shuster 1963), becomes fully operative
at later stages (Ashihara 1983), and (2) ATP and GTP pro-
duced by the de novo and salvage pathways of germinating
Plant nucleotide metabolism 1281
embryos appear to occur as two separate pools with little or
no crossover (Anderson 1979), and they possibly also have
distinct functions.
The third phase of the metabolic pattern of purine metabo-
lism, following the salvage phase, is the formation of ureides,
which represent an important nitrogen source during the early
phases of germination, particularly in leguminous species
(Thomas and Schrader 1981). As suggested by Fujiwara and
Yamaguchi (1978), the main pathway for allantoin and allan-
toic acid formation is possibly by degradation of purines (see
Fig. 5 A). This concept is further supported by the observation
that the degradation pathway of purine nucleotides, as esti-
mated by hypoxanthine and inosine catabolism is also oper-
ative during germination. In black gram seedlings, for exam-
ple, almost 60 % of radioactivity from hypoxanthine was incor-
porated into the ureide fraction after a few days of germina-
tion (Ashihara and Nobusawa 1981). Extensive degradation of
this precursor was possibly due to the low activity of HPRT
compared to that of other salvage enzymes (Nobusawa and
Ashihara 1983). The presence of this enzyme during germina-
tion has also been documented in wheat (Price and Murray
1969) and lupin (Guranowski and Barankiewicz 1979) seeds.
In white spruce somatic embryos, a conspicuous formation of
ureides via the purine degradation pathway was only ob-
served after 4 days in germination (Stasolla et al. 2001b).
Another important consideration when conducting studies
on purine metabolism during germination is the possibility
that purines released from catabolic events in specific re-
gions of the embryo are re-metabolised in other parts of the
embryo. In germinating wheat embryos, for example, exten-
sive RNA and nucleotide catabolism, as well as high 3′-
nucleosidase activity, has been found to increase at germina-
tion, especially within the coleorhiza (Shuster and Gifford
1962, Price and Ey 1969). Price and Murray (1969) further
demonstrated that these degradation products generated in
the coleorhiza are salvaged by root and leaf tissue of the em-
bryos. An analogous case of purine transport between cotyle-
dons and embryonic axis has been documented in black
gram embryos (Ashihara 1983).
As is true of purine nucleotide metabolism, changes in py-
rimidine metabolism delineate defined stages of embryo ger-
mination. Although both the de novo and salvage pathways
(see Figs. 2 and 4 B) are operative at germination, their con-
tribution to the enlargement of the nucleotide pool during the
process appears to be tightly regulated. As reported by Ashi-
hara (1977) and Stasolla et al. (2001 c), metabolism of pyrimi-
dines, in fact, can be divided in two distinct phases: (1) «sal-
vage synthesis», at the inception of germination, and (2) «de
novo synthesis», observed at later stages of germination.
Active production of pyrimidine nucleotides following imbi-
bition occurs via the salvage pathway. In black gram cotyle-
dons, for example, utilization of uracil and uridine for RNA
and nucleotide biosynthesis increased markedly during the
first 24 h of germination, before declining at later stages
(Ashihara 1977). A similar result was also reported in germi-
1282 Claudio Stasolla et al.
nating white spruce somatic (Stasolla et al. 2001c) and zygo-
tic (Stasolla et al. 2002) embryos. In somatic embryos, for
example, uridine incorporation into nucleic acids and nucleo-
tides was high in dried embryos, and it slowly declined during
the first 6 days of germination (Stasolla et al. 2001 c). In this
study, salvage of uridine was mainly catalysed by UK, and to
a lesser extent by non-specific phosphotransferases, whose
activities were also observed during germination of maize
(Zea mays) seeds (Wanka and Walboomers 1966) and wheat
grains (Mazus and Buchowicz 1972).
As germination progresses, the contribution of the salvage
pathway to the overall pyrimidine nucleotide pool declines.
The mechanism limiting the activity of salvage synthesis dur-
ing the later stages of germination has not been fully eluci-
dated, although conversion of UMP to UDP may represent the
limiting step. In pea (Pisum sativum) cotyledons incubated
with labeled uracil and uridine, for example, more radioactiv-
ity was recovered in the UMP fraction than in the UDP-UDPG
fraction (Ross et al. 1971). Alternatively, the rate of conversion
of pyrimidine bases and nucleosides into nucleotides may be
determined by the activity of the relative salvage enzymes. In
germinating white spruce somatic and zygotic embryos, for
example, a decrease in uridine salvage paralleled a pro-
nounced decline in UK activity (Stasolla et al. 2001c, 2002). A
general picture emerging from germination studies is that uri-
dine is a more rapidly utilized substrate for the synthesis of
salvage products than uracil, as a large percentage of uracil is
often degraded through a reductive catabolic pathway (Evans
and Axelrod 1960, Ashihara 1977, Stasolla et al. 2001c).
The decreased salvage activity as germination progresses
is accompanied by an increased activity of the de novo path-
way. Utilization of orotic acid for nucleotide and nucleic acid
synthesis, in fact, was found to increase in cotyledons of ger-
minating black gram (Ashihara 1977) and Alaska pea seeds
(Ross et al. 1971), as well as in germinating white spruce so-
matic embryos (Stasolla et al. 2001 c). The restoration of the
de novo pathway seems to be critical for ensuring that the
embryos have sufficient nucleotides to support active nucleic
acid synthesis. Increasing levels of nucleotides were demon-
strated during germination of both pine pollen tubes (Ny-
gaard 1973) and bacterial spores (Setlow and Kornberg
1970).
The restoration of the de novo machinery as germination
progresses may be due to the increased activity of several
enzymes involved in the de novo pathway, including OPRT,
which, in concert with OMP decarboxylase, catalyzes the
conversion of orotic acid to UMP. In support of this concept is
the observation that the activity of OPRT is generally low at
the beginning of germination but increases at later stages
(Ross et al. 1971, Ross and Murray 1971, Stasolla et al.
2001 c). Besides OPRT, the levels of other enzymes involved
in the construction of the pyrimidine ring, including carbamoyl
phosphate synthetase and aspartate carbamoyltransferase,
have been found to increase as germination progresses (Ma-
zus and Buchowicz 1972). An alternative explanation for the
restored de novo synthesis of pyrimidine nucleotides has
been proposed by Brown and Wray (1968), who observed in-
creased activity of the pentose phosphate pathway over the
first 6 days of germination in Meteor pea cotyledons. This
pathway is responsible for the production of ribose-5-phos-
phate, substrate for the synthesis of PRPP, which is required
for the conversion of orotic acid to UMP.
Studies on deoxyribonucleotide also revealed that both de
novo and salvage pathways are operative during germina-
tion. In germinating white spruce somatic embryos, the activ-
ity of the de novo pathway was demonstrated by the low, but
significant utilization of supplied cytidine for DNA synthesis.
Conversion of this precursor to CMP was mainly regulated by
the activity of cytidine kinase (CK), and to a lesser extent, by
non-specific phosphotransferases (Stasolla et al. 2003 a). The
role played by CK in cytidine metabolism at germination was
also demonstrated in corn seedlings (Wanka and Bauer
1967).
A large fraction of cytidine was also incorporated into the
RNA fraction. In germinating white spruce somatic embryos,
utilization of cytidine for RNA synthesis has been demon-
strated to occur directly, after conversion to CMP, or indirectly,
via deamination to uridine and ammonia by deaminase activ-
ity (Stasolla et al. 2003 a). The presence of this latter route in
plant cells was long ago demonstrated by Ross and Cole
(1968).
Salvage synthesis of deoxyribonucleotides at germination
was also demonstrated by following the metabolic fate of de-
oxycytidine and thymidine (Wanka and Walboomers 1966,
Kameyama et al. 1985, Stasolla et al. 2002, 2003 a). In germi-
nating white spruce somatic embryos, utilization of deoxycyti-
dine for DNA synthesis was higher than that measured in fully
matured embryos (Stasolla et al. 2003 a). In this study, it was
also demonstrated that both deoxycytidine kinase (dCK) and
non-specific phosphotransferases contributed equally to the
salvage of deoxycytidine.
Compared to deoxycytidine, a large fraction of supplied
thymidine is degraded to CO2 during germination (Kame-
yama et al. 1985, Stasolla et al. 2002). Increasing utilization of
this precursor for nucleic acid synthesis was observed in ger-
minating black gram seeds (Kameyama et al. 1985), as well
as in germinating white spruce zygotic embryos (Stasolla et
al. 2002). In white spruce somatic embryos, thymidine sal-
vage appears to be critical for post-embryonic growth, as
experimental manipulations of the culture conditions that
decrease the germination frequency of the embryos also
result in a decrease of thymidine anabolism (Stasolla 2001).
Conversion of thymidine to dTMP is mediated by thymidine
kinase (TK), as well as by non-specific phosphotransferases.
The contribution of these two enzymes to the amount of thymi-
dine being salvaged appears to be species dependent.
Wanka and Walboomers (1966) reported the activity of thymi-
dine kinase in maize seedlings, however, it has been indi-
cated that thymidine kinase measured in crude extracts may
be due to combined activities of the nucleoside phospho-

transferases and ATP phosphohydrolase (Mullin and Fites
1978). The activity of thymidine kinase, was not detected in
germinating black gram embryos (Kameyama et al. 1985);
however, relatively high activity of thymidine kinase was
observed in both somatic (Stasolla et al. 2003 a) and zygotic
(Stasolla et al. 2002) germinating embryos of white spruce.
3.3 In vitro organogenesis
The only two studies available on nucleotide biosynthesis
during in vitro organogenesis are those dealing with shoot
bud formation from epicotyls of white spruce and excised
cotyledons of radiata pine (Stasolla et al., unpublished data).
Investigations on both purine and pyrimidine metabolism
indicate that the production of nucleotides via the salvage
synthesis plays an important role during the process of shoot
formation. In white spruce, epicotyls cultured under shoot-
forming (SF) conditions more actively utilize adenine and
adenosine for nucleotide and nucleic acid synthesis, com-
pared to those cultured on non-shoot forming (NSF) medium.
This higher salvage synthesis, mainly due to increased activ-
ity of APRT and AK, occurs after days 10 in culture, when
shoot formation is initiated. The importance of the salvage ac-
tivity during the organogenic process was also demonstrated
in excised cotyledon explants of radiata pine, where an in-
creased incorporation of uracil into the nucleic acid fraction,
as well as a higher activity of the uracil salvage enzyme, ura-
cil phosphoribosyltransferase (UPRT), was observed during
the formation of shoot primordia (Stasolla, Loukanina, Ashi-
hara, Yeung, Thorpe, unpublished data).
3.4 Breaking bud dormancy
In various plants, a dormant period is observed during the
vegetive cycle. The resting state that generally occurs during
winter is broken by low temperature. These plants recover
their growth potentialities in the following spring. In ash (Fraxi-
nus excelsior), and peach (Prunus persica) trees, and straw-
berry (Fragaria × ananassa) plants, breaking of dormancy is
accompanied by an increased capacity of the salvage path-
ways of adenosine and adenine (Barnola et al. 1986, Balan-
dier et al. 1993, Robert and Petel 2001). During the dormancy
breaking of buds, an increase in the levels of nucleoside tri-
phosphates, such as ATP and GTP, has been observed after
treatment with adenosine. This is referred to as the «nucleo-
tide test» (Lavarenne et al. 1982, Robert et al. 1997). Dor-
mancy breaking is accompanied by an increase in the activity
of adenosine kinase, adenine phosphoribosyltransferase and/
or adenosine nucleosidase (Faye and Le Floc’h 1997, Robert
and Petel 2001).
3.5 Fruit ripening
Changes in the activity of nucleotide biosynthesis during de-
velopment of orange (Citrus sinensis) fruits were estimated
Plant nucleotide metabolism 1283
using various 14C-labeled precursors (Tomlinson and Lovatt
1987). Pyrimidine biosynthesis de novo in whole navel orange
fruits increased during the cell-division phase of fruit develop-
ment. Capacity of peel tissue to synthesise pyrimidine nu-
cleotides de novo decreased with fruit development up to 5
months. In contrast, synthesis of purine nucleotides was not
related to any specific stage of fruit development.
3.6 Storage organ development and sprouting
Studies on the enzymes and general metabolism of nucleoti-
des in storage organs, such as roots and tubers, have been
carried out mainly with tubers of Jerusalem artichoke (Heliant-
hus tuberosus) and potato (Solanum tuberosum). The key en-
zymes of purine nucleotide metabolism, AMP deaminase (Le
Floc’h and Lafleuriel 1983 a) and guanylate kinase (Le Floc’h
and Lafleuriel 1990), were purified, and their regulatory pro-
perties and subcellular localization were investigated. Gend-
raud (1975) reported that dormant Jerusalem artichoke buds
had adenosine salvage activity. However, conversion of sal-
vaged adenine nucleotides to other nucleotides appeared
only in non-dormant ones. When non-dormant Jerusalem
artichoke tubers were transferred to a temperature high
enough to allow growth, adenosine nucleosidase activity in-
creased (Le Floc’h and Lafleuriel 1984).
In potato tubers, nucleotide-sugars, such as ADP-glucose
and UDPG, play an important role in starch formation and
sugar-starch conversion. Recently, Loef et al. (1999, 2001) ar-
gued that increased levels of pyrimidine and purine nucleoti-
des formed from exogenously supplied orotate and adenine
stimulate the conversion of sucrose to starch. Detailed stud-
ies on purine and pyrimidine metabolism in potato tubers
have been recently conducted by Katahira and Ashihara
(2002).
(a) Tuber growing stage
In actively growing potato tubers, in situ activities of the de
novo pathway of purine and pyrimidine nucleotide synthesis
estimated from the incorporation of radioactivity from
[14C]formate, [2-14C]glycine, [2-14C]orotate (see Figs. 1 and 2)
were extremely high. Furthermore, activities of the salvage
pathways of adenine, adenosine, uridine and cytidine esti-
mated by the metabolic fate of labeled precursors and activ-
ities of respective enzymes, i.e., adenine phosphoribosyl-
transferase, AK and UK/cytidine kinase (see Fig. 4) were also
high in this stage. This high salvage activity in growing tubers
may be due to rapid turnover of nucleotides, and it would
contribute to the maintenance of sufficient energy and build-
ing blocks required for cell division and enlargement, as well
as starch synthesis. Although to date no evidence is avail-
able, it is possible that some nucleobases and nucleosides
produced in leaves may be translocated to growing tubers. If
so, the active salvage pathways observed in growing tubers
are a very efficient mechanism to make nucleotides without
costs.
1284 Claudio Stasolla et al.
Metabolism of deoxyribonucleotides (see Fig. 3) in growing
tubers was investigated using [2-14C]cytidine, [2-14C]deoxy-
cytidine and [2-14C]thymidine. Both de novo synthesis of
deoxyribonucleotides and salvage synthesis from deoxyribo-
nucleosides were operative in potato tubers. These activities
may contribute to the active synthesis of DNA in this stage.
Rapid degradation of exogenously supplied [8-14C]inosine
and [2-14C]uracil indicated that activity (enzyme machinery)
of the degradation pathways of purine and pyrimidine nucleo-
tides to CO2 and NH3 (see Fig. 5) was present in the growing
tubers. However, 14C-labelled adenine, adenosine and uridine
were exclusively salvaged to nucleotides and nucleic acids
and only small amounts of [8-14C]adenosine and [2-14C]uridine
taken up during 2 h-pulse incubation were degraded even
after a 12 h-chase. Therefore, it appears that little inosine and
uracil were produced in growing tubers; as a result, degrada-
tion of purine and pyrimidine skeletons is very limited.
In growing potato tubers, a novel deoxycytidine salvage
pathway, viz., deoxycytidine → deoxyuridine → uridine → UMP
has been proposed to occur (Katahira and Ashihara 2002).
(b) Mature (Dormant) stage
Translocation of assimilates in leaves to storage organs stops
shortly after leaf senescence begins, and tuber growth stops.
In the dormant tubers, activities of the de novo pathways of
purine and pyrimidine biosynthesis decreased, while different
trends were observed in the salvage pathways. In contrast to
pyrimidine salvage activities, i.e., nucleotide synthesis from
uridine and cytidine, which were as markedly reduced as
those of de novo pathways, adenosine and adenine salvage
activities decreased only slightly. Catabolic activity of nucleo-
tides was significantly reduced in the dormant tubers; and
this may reflect the low turnover of nucleotides at this stage.
(c) Sprouting stage
During the subsequent sprouting period, constituents of tu-
bers are used for the growth of buds. In tubers, activities of
the de novo and salvage pathways of purine and pyrimidine
biosynthesis were similar to those in the mature stage; the
only exception was the increase in the in situ uridine salvage
activity. In addition to uridine kinase, non-specific nucleoside
phosphotransferase may contribute to this salvage, as
judged by the finding that the latter enzyme activity doubled
during the sprouting period. UDPG content, a rate-limiting
factor for the conversion of starch to sucrose (Sowokinos et
al. 1993), may be mainly dependent on the uridine salvage
activity at this stage.
Although little work has been performed on the transport of
metabolites of nucleotide metabolism in plants, transport from
organ to organ seems to be carried out mainly at the nucleo-
side and nucleobase levels (see Ashihara and Crozier 1999).
For example, in germinating castor beans (Ricinus commu-
nis), adenosine, guanosine, inosine and adenine were trans-
ported from the endosperm to the cotyledons (Kombrink and
Beevers 1983). In potato tubers, detailed changes in the lev-
els of nucleosides and nucleobases during the sprouting
stage have not yet been determined. However, [2-14C]uridine,
[2-14C]cytidine and [2-14C]uracil injected into the tubers at
this stage were transported to the sprouts and mainly recov-
ered as nucleic acids after 3 days.
In sprouts, the activities of both the de novo and salvage
pathways of purine and pyrimidine nucleotides were high
(Katahira and Ashihara 2001). Tremendously elevated levels
of all participating enzymes of the biosynthetic pathways
were observed. Among them, the activity of adenosine nu-
cleosidase was 650-fold higher than that in the tubers. Le
Floc’h and Lafleuriel (1984) also found that increased adeno-
sine nucleosidase activity accompanied the dormancy re-
lease in tubers of Jerusalem artichoke. The role of adenosine
nucleosidase in plants has not yet clearly been elucidated.
This enzyme also acts as a hydrolase of cytokinin nucleosi-
dase (Chen and Kristopeit 1981), so its activity may be related
to cytokinin metabolism. In support of this notion, the concen-
trations of total cytokinin in the buds of potato tubers in-
creased up to 50-fold during dormancy break (Turnbull and
Hanke 1985).
3.7 Leaf senescence
The pattern of uracil metabolism during senescence of to-
bacco leaves has been documented by Ashihara (1981), who
observed extensive uracil salvage activity. Approximately
60 % of uracil was converted to nucleotides and further met-
abolised to UDPG and nucleic acids. This high salvage activ-
ity was retained in the early senescent stage of leaves, when
chlorophyll content decreased to nearly half of its original
level. A slight decrease in uracil utilization occurred in older
leaves, and this was due to a decreased activity of uracil
phosphoribosyltransferase and phosphoribosylpyrophos-
phate synthetase. Uracil was degraded to CO2 via β-ureido-
propionate. This degradation activity was almost constant
(approx. 30 – 35 % of uracil taken up by the leaf segments) at
different stages of leaf senescence. However, in the most se-
nescent leaf, nearly half of uracil taken up remained unmeta-
bolised.
3.8 Cultured plant cells
Suspension cultured cells have represented a valuable tool
for studying growth and differentiation of plant cells. In the
past few years, this system has been successfully employed
for investigating changes in nucleotide synthesis and utiliza-
tion during synchronous growth of cultured cells (reviewed by
Wagner and Backer 1992). Inoculation of starved cells into
fresh medium, in fact, results in synchronous growth, as well
as synchronous uptake and utilization of metabolites (King
and Street 1977, Wylegalla et al. 1985). Cells cultured under

these conditions display very similar growth behaviour, which
can be divided into (1) lag phase, (2) cell division phase,
characterized by an increase in cell mass, (3) cell elongation
phase caused by increased vacuolation, and (4) stationary/-
starvation phase, delineated by a decreased availability of
nutrients in the medium. Defined morphological and meta-
bolic events occur in each phase (see Wagner and Backer
1992).
(a) Changes of nucleotide pools during cell growth
Biochemical studies conducted on synchronously growing
cultured cells in several plant species have revealed that the
concentration of nucleotides in a particular phase may repre-
sent a regulatory signal affecting the transition of the cells to
the next phase (Meyer and Wagner 1985 a, Wylegalla et al.
1985, Wagner and Backer 1992). Following inoculation of
starved cells into fresh medium a pronounced increase in nu-
cleotide levels has been observed in many species, including
sycamore (Acer pseudoplatanus) (Brown and Short 1969,
Shimizu et al. 1977), Vinca rosea (= Catharanthus roseus)
(Shimazaki et al. 1982, Sasamoto and Ashihara 1988), soy-
bean (De Klerk-Kiebert and Van der Plas 1984), and Datura
innoxia (Meyer and Wagner 1985 a). A similar, but less pro-
nounced increase was also observed for guanine nucleotides
(Meyer and Wagner 1985 a). Increased nucleotide biosynthe-
sis at the lag phase seems to be a prerequisite for the initia-
tion of the proliferative phase, where intense DNA and RNA
synthesis occurs (Wylegalla et al. 1985). In cultured cells of
Datura innoxia, for example, the intracellular increase of nu-
cleoside triphosphates is similar, but slightly precedes that of
the RNA content, which reaches its maximum in the cell divi-
sion phase (Wylegalla et al. 1985).
During cell division, the nucleotide pool decreases, as well
as the energy charge (ATP + 0.5 ADP)/(ATP + ADP + AMP),
possibly because of ATP consumption. The energy charge, in
agreement with respiratory activity data (Givan and Collin
1967), usually increases in the first hours of the lag phase,
and then it decreases during the cell division phase, before
attaining constant levels in the following cell-elongation and
starvation phases (Shimizu et al. 1977, Shimazaki et al. 1982,
Meyer and Wagner 1985 a). Based on these results, Shima-
zaki et al. (1982) divided the adenylate metabolism of Catha-
ranthus roseus cells into an «energy generating stage» dur-
ing the lag phase, and an «energy utilizing stage» which is
initiated with the cell division stages. In cultured cells of to-
bacco (Nicotiana tabacum) and soybean, however, no de-
crease in energy charge was observed during cell division
(DeKlerk-Kiebert and Van der Plas 1984). In the final elonga-
tion and starvation phases, the overall nucleotide pool attains
a low, but constant value. Meyer and Wagner (1985 a) sug-
gest that at this stage, as no anabolic consumption occurs,
nucleotides are most likely degraded to nucleosides. Salvage
of these nucleosides upon inoculation into fresh medium will
be discussed later.
Plant nucleotide metabolism 1285
Qualitative differences within the overall nucleotide pool
also seem to exist during growth of cultured cells. A compar-
ison of the different components of the nucleotide pool
among different cell culture systems, including sycamore
(Brown and Short 1969), Datura (Meyer and Wagner 1985 a,
Mitsui and Ashihara 1988), and tobacco (Meyer and Wagner
1985 b), reveals uniform results. In general, the lower fraction
was composed by cytosine nucleotides (less than 3 %), and
the highest by uracil nucleotides (more than 50 %), with the
UDP-sugars as the main fraction (see Wagner and Backer
1992). The high proportion of UDP-sugars observed within
the total nucleotide pool, as well as their increased content at
the inception of the cell division phase may be required for
cell-wall production. Further studies on the relative size of the
nucleotide pools also revealed a constant uracil / adenine nu-
cleotide ratio during the entire growth curve of Datura (Meyer
and Wagner 1985 a) and tobacco (Meyer and Wagner 1985 b)
cells. This constant ratio was also observed in cells of an au-
xotrophic mutant of Datura innoxia, which needed supple-
mentation of adenine, adenosine, or inosine for normal growth
(Mitsui and Ashihara 1988). Wagner and Backer (1992) spec-
ulated that metabolic adjustments of the uracil and adenine
nucleotide pools denote the presence of regulatory mecha-
nisms controlling the endogenous levels of purines and pyri-
midine nucleotides. Regulation of these pools has been re-
vealed to be critical for normal growth and development, as
reported in animal and bacterial systems (Chou et al. 1984,
Shimosaka et al. 1984).
(b) Synthesis of nucleotides during cell growth
As reported above, a major synthesis of nucleotides occurs
during the lag phase of cell growth, in preparation for cell pro-
liferation. By measuring nucleotide levels in Datura cells
every 6 h after inoculation into fresh medium, Meyer and Wag-
ner (1985 a) demonstrated a biphasic behavior in the increase
of the overall nucleotide pool. For both adenine and uracil nu-
cleotide pools the first increase was observed at 6 h and 15 h
respectively, whereas a second increase occurred a few days
later. A similar behavior during the lag phase was also ob-
served for the uptake of inorganic phosphate. (Wylegalla et
al. 1985). These results, as interpreted by Kanamori-Fukuda
et al. (1981), suggest that nucleotide synthesis during cell
growth in suspension culture can be divided in two stages: (1)
a «turn-over or salvage phase» during the lag phase, and (2)
a «true biosynthetic stage», characterized by the reactivation
of the de novo pathway, at the inception of cell division. This
concept was substantiated by tracer experiments. Pyrimidine
salvage (see Fig. 4 B), as estimated by the incorporation of
uracil and uridine into the RNA fraction (Kanamori et al. 1979,
Kanamori-Fukuda et al. 1981), as well as salvage of purine
precursors (see Fig. 4 A), including adenine, adenosine, gua-
nine, and hypoxanthine (Shimazaki and Ashihara 1982, Hi-
rose and Ashihara 1984), was high during the lag phase of
cultured cells and decreased in the following stages. This ex-
1286 Claudio Stasolla et al.
tensive utilization of bases and nucleosides for nucleotide
synthesis was mainly ascribed to the high activities of the
major purine and pyrimidine salvage enzymes (Kanamori et
al. 1979, Kanamori-Fukuda et al. 1981, Hirose and Ashihara
1984). Activation of the de novo pathway appears to be a
later event. In cultured Catharanthus roseus cells, orotic acid
utilization for RNA synthesis increased markedly during the
cell division phase (Kanamori et al. 1979). This delay in the
restoration of the de novo machinery is likely due to a lag in
the reactivation of enzymes participating in the de novo path-
way of both purines and pyrimidines. Activities of PRPP syn-
thetase and pyrimidine enzymes, such as carbamoylphos-
phate synthetase (step 1, Fig. 2) and OPRT (step 5, Fig. 2),
were highest in the cell division phase in Catharanthus roseus
cells (Kanamori et al. 1979, Kanamori-Fukuda et al. 1981). The
activities of purine enzymes involved in the de novo pathway,
including glycinamide ribonucleotide synthetase (step 2,
Fig. 1) and amido phosphoribosyltransferase (step1, Fig. 1)
showed a similar pattern in cultured carrot cells (Ashihara
and Nygaard 1989).
Although much work on nucleic acid metabolism during
cell growth has been conducted in flowering plants, the activ-
ities of de novo and salvage pathways have been demon-
strated only recently in cultured cells of a coniferous species,
Picea glauca (Ashihara et al. 2000).
(c) Regulation of cell growth by nucleotide availability
Studies conducted on uptake and utilization of Pi have dem-
onstrated that cellular phosphate may act as the signal for the
initiation and termination of cell division, possibly by regulat-
ing the availability of nucleoside triphosphates within the sys-
tem (Wylegalla et al. 1985, Ukaji and Ashihara 1986, Wagner
and Backer 1992). In Catharanthus and Datura cells, the up-
take of phosphate from the medium is completed within the
lag phase. At the beginning of cell proliferation, the phos-
phate pool is consumed and this parallels a decrease of the
cellular nucleoside-triphosphate pool (Wylegalla et al. 1985,
Ukaji and Ashihara 1986). Depletion of intracellular phos-
phate and nucleoside triphosphates below a threshold may
represent a metabolic switch, which terminates cell division
and initiates cell elongation (Wylegalla et al. 1985, Wagner
and Backer 1992). However, it should be mentioned that Pi
may still be found in the vacuole after the medium is phos-
phate-free (Reibelle et al. 1983).
Studies on nucleotide metabolism during the stationary
phase have been mainly conducted in Datura innoxia cells,
where the length of this phase can be extended up to two
weeks, without affecting cell viability (Wylegalla et al. 1985).
In general, the stationary phase shows a reduction of both
DNA and RNA (Wylegalla et al. 1985, Ukaji and Ashihara
1986, Ashihara and Nygaard 1989), which is probably the re-
sult of a decreased nucleotide pool. It is also interesting to
notice that the overall energy charge of the nucleotide pool is
generally high (see Wagner and Backer 1992). As suggested
by Reibelle et al. (1983), this high ratio may be a metabolic
condition for survival.
(d) Effect of phosphate on nucleotide metabolism
Cultured plant cells have been largely utilized for detailed ex-
amination on the metabolic role of nutrients. Among the many
inorganic nutrients, inorganic phosphate (Pi) has been shown
to participate in many metabolic processes affecting plant
cell growth and development (see Wagner and Backer 1992).
Complete uptake of Pi by cultured cells is generally achieved
during the first day after inoculation into fresh medium (Wyle-
galla et al. 1985), although immediate uptake was reported in
Catharanthus roseus cells grown in the absence of Pi for a
67h period (Ashihara and Ukaji 1986).
Overall changes in cellular metabolism have been ob-
served as a result of Pi starvation. These include reduced
rates of respiration (Li and Ashihara 1990), decreased protein
and nucleic acid synthesis (Ukaji and Ashihara 1987, Li and
Ashihara 1989), development of Pi-deficient inducible bypas-
ses of respiration (Duff et al. 1989, Nagano and Ashihara
1993, Nagano et al. 1994), as well as changes in nucleotide
pool size and composition (Ukaji and Ashihara 1987, Dancer
et al. 1990, Li and Ashihara 1990, Shimano and Ashihara
2001). In Catharanthus roseus cells, for example, the endoge-
nous ATP level of Pi-starved cells was less than 35 nmol g
FW –1. Re-inoculation of these cells with 10 – 20 mmol/L Pi, re-
sulted in a dramatic increase in ATP level (180 nmol g FW –1)
(Ashihara and Ukaji 1986). A similar result was also docu-
mented by Dancer et al. (1990), who observed a reduction in
the overall adenine and uracil nucleotide pools, as well as a
decrease in the ATP/ADP and UTP/UDP ratios in Chenopo-
dium rubrum cell suspension cultures grown in the absence
of Pi. These results, together with the inability of cells inocu-
lated into a Pi-free medium to increase their endogenous level
of ATP (Ukaji and Ashihara 1986), clearly suggest that the rise
in nucleotide content observed during the lag phase, that is
necessary for the subsequent cell division phase, is strictly
dependent upon Pi availability in the medium (see Wagner
and Backer 1992).
The effects of Pi-availability on the de novo and salvage
synthesis of nucleotides were investigated by Ashihara et al.
(1988). Comparative studies on nucleotide synthesis between
Catharanthus roseus cells grown in the presence or absence
of Pi, revealed increased activities of de novo and salvage
pathways of both purines and pyrimidine nucleotides in the
former. In the same study, it was also demonstrated that the
rate of uridine degradation was significantly reduced in cells
grown in the presence of Pi. Although the role played by Pi
during the synthesis of nucleotides needs clarification, sev-
eral studies indicate that the activities of both de novo and
salvage pathways are regulated by PRPP concentration
which was increased by Pi. Hirose and Ashihara (1983 b) re-
ported that the level of PRPP was very low in the stationary
phase of Catharanthus roseus cells in culture, but increased

rapidly after inoculation into fresh medium. This increase,
however, was not observed if the cells were transferred into a
medium devoid of Pi (Ukaji and Ashihara 1987).
3.9 Environmental factors
(a) Effects of salts on nucleotide biosynthesis
Plants require some salts for growth, but concentrations
above the optimum, often result in reduced growth and yield.
Expenditure of energy derived from oxidation of carbohy-
drates and salt pumping is often observed in salt-stressed
plants (Nieman et al. 1988). Changes in the synthesis and uti-
lization of nucleotides, especially ATP, in response to salt
stress have been reported in several systems, including
maize, pepper (Capsicum annuum), safflower (Carthamus
tinctorius), and mangrove (Sonneratia alba) (Peterson et al.
1987, 1988, Nieman et al. 1988, Akatsu et al. 1996). From
these studies, it emerges that salt stress decreases the total
nucleotide pool, but increases the adenylate energy charge.
In mangrove, for example, the energy charge of cells cultured
in the presence of 100 mmol/L NaCl was 0.82, compared to
0.72 of control cells (Akatsu et al. 1996). The result, similar to
that described by Kubota and Ashihara (1993) in Catharant-
hus roseus cells, indicates the presence of an active adeny-
late kinase, which is responsible for the enlargement of the
endogenous ATP pool. High levels of ATP may be necessary
for removal of NaCl from the cytosol of cells to the outside of
the cells and/or to the vacuoles. Tracer experimental studies,
performed by Akatsu et al. (1996) further demonstrated that in
NaCl-treated cultured cells of mangrove a low purine degra-
dation activity allows the accumulation of bases and nucleo-
sides to be salvaged for nucleotide synthesis. This strategy
would represent an efficient adaptation to salt stress (Akatsu
et al. 1996).
Ashihara et al. (2003) investigated adenosine metabolism
in leaves of various mangrove plants. The activity of adeno-
sine salvage was extremely high in the typical mangrove
plants, Rhizophora mucronata, Burugiera gymnorrhiza, Kan-
delia candel and Sonneratia alba. In these mangrove plants,
up to 90 % of [8-14C]adenosine taken up by leaf segments
was converted to adenine nucleotides. In contrast to the
mangrove plants mentioned, which contain high levels of
sugar alcohols as compatible solutes, Avicennia marina,
which accumulates glycinebetaine, showed a different pat-
tern of adenosine metabolism. The glycinebetaine biosyn-
thetic pathway from phosphoethanolamine includes three
methylation steps that utilize S-adenosylmethionine (SAM) as
the methyl donor. In this process SAM is converted to S-ade-
nosylhomocysteine (SAH), which in turn is hydrolysed to ho-
mocysteine and adenosine. SAH hydrolase catalyzes both
the synthesis and hydrolysis of SAH. As SAH is a potent in-
hibitor of the N-methyltransferase reactions, removal of SAH
is essential to maintain the biosynthesis of glycinebetaine.
Plant nucleotide metabolism 1287
Probably for this reason, very active adenosine nucleosidase
is present in A. marina, and 50 – 60 % of exogenously sup-
plied [8-14C]adenosine was recovered as adenine. Com-
pared to ordinary biomass trees, such as poplar (Populus
alba), activity of adenosine salvage is extremely high in most
mangrove trees, which may act as a very efficient ATP regen-
erating system. As mangrove plants can produce large
amounts of ATP to facilitate the removal and/or transport of
salts into vacuoles and to synthesize compounds that func-
tion as compatible solutes to adjust the osmotic potential of
their tissues, adenosine recycling may be very important. Fur-
thermore, high adenosine nucleosidase activity in glycinebe-
taine-producing mangrove plants may be related to the con-
tinuous supply of methyl donors for biosynthesis of the com-
patible solute.
Recent work indicates that the activation of adenosine ki-
nase and adenosine nucleosidase may participate in these
processes, in response to salt stress Weretilnyk et al. 2001,
Suzuki et al. 2003). Weretilnyk et al. (2001) observed that in
salt-stressed spinach (Spinacia oleracea) and sugar beet
(Beta vulgaris), adenosine kinase activity, as well as AK pro-
tein and transcript accumulation increased. This increase
was related to the synthesis of glycinebetaine in response to
high salt concentration. In general, removal of adenosine
seems to be necessary to sustain the SAM dependent meth-
yltransferase activity. In plant cells adenosine deaminase is
almost absent (see Section 2.8), as a result, adenosine kinase
and/or adenosine nucleosidase have important roles for this
purpose (Ashihara and Crozier 2001, Koshiishi et al. 2001,
Moffatt and Weretilnyk 2001, Moffatt and Ashihara 2002,
Suzuki et al. 2003).
(b) Effect of water stress
Alternation of regulation of pyrimidine biosynthesis by water
stress has been suggested in ragi (Eleusine coracana) seed-
lings. Feedback inhibition of aspartate transcarbamoylase
(step 2, Fig. 2) by UMP found in control plants disappeared in
water-stressed seedlings (Kandpal and Rao 1984). In some
proteins, water stress caused a change in the ratio of – SH/S-
S bonds. Therefore, a similar change might lead to the desen-
sitization of the regulatory properties of aspartate transcarba-
moylase. The physiological meaning of this change has not
yet been elucidated.
(c) Effect of iron-deficiency
In Fe-deficient Graminaceous plants, production of the mugi-
neric acid family phytosiderophores (MAs), which solubilize
inorganic Fe(III) compounds by chelating is increased. Simul-
taneously, expression of adenine phosphoribosyltransferase
gene and adenine phosphoribosyltransferase activity devel-
oped remarkably by Fe-deficiency (Itai et al. 2000). As MAs
are produced from SAM, and adenine is released from the
methionine cycle for regeneration of SAM, adenine recycling
by adenine phosphoribosyltransferase for ATP synthesis
1288 Claudio Stasolla et al.
seems to be functional for maintenance of a sufficient supply
of ATP for SAM generation.
(d) Effect of light
Early studies described the effects of light on RNA synthesis
through the de novo and the salvage pathways of pyrimidine
synthesis. In cucumber (Cucumus sativus) seedlings, incor-
poration of radioactivity from [6-14C]orotate, but not [6-
14
C]uracil, into RNA pyrimidine bases was stimulated by the
light (Šebesta et al. 1964).
The effect of brief light illumination by microbeams on the
adenine nucleotide levels in maize primary roots has been in-
vestigated. Total adenylate pool was increased up to 150 % of
the initial value within 2 min (Pilet and Gabathuler 1982). Fur-
ther studies are needed to elucidate the mechanism of these
light effects on nucleotide metabolism.
4. Molecular studies on purine and
pyrimidine nucleotides
Despite the extensive information dealing with physiological
and biochemical events associated with purine and pyrimi-
dine nucleotide biosynthesis in plants, published information
regarding the molecular mechanisms regulating these path-
ways is very limited. It is only in recent years, through the
combined application of novel molecular and genetic techni-
ques, that the function of several enzymes involved in synthe-
sis and degradation of nucleotides has been established at a
molecular level, and mainly from studies carried out with Ara-
bidopsis mutants (reviewed by Moffatt and Ashihara 2002).
4.1 Purine nucleotide synthesis and degradation
Since PRPP is a fundamental precursor of the de novo and
salvage synthesis of both purine and pyrimidine nucleotide
(Figs. 1 and 2), it is not surprising that all living organisms
have at least one gene encoding PRPP synthetase (see Mof-
fatt and Ashihara 2002). Through complementation studies in
bacterial cells lacking PRPP synthetase activity, Krath and
Hove-Jensen (1999) isolated four PRPP synthetase cDNAs
from spinach tissue. By a combined application of kinetic
investigations and phylogenetic comparisons, the authors
demonstrated that two of the four cDNAs identified may rep-
resent a novel class of PRPP synthetase.
In animals, amido phosphoribosyltransferase (ATase, step
1, Fig. 1) is an important enzyme involved in the first step of
the de novo purine nucleotide biosynthesis (see Moffatt and
Ashihara 2002). Two ATase sequences, designated as AtA-
Tase1 and 2, were obtained from the Arabidopsis data base
(Genebank accessions D28868 and D28869) (Ito et al. 1994).
Expression of AtATase1 is preferential in young floral buds,
whereas AtATase2 expression is highest in flowers and roots
(see Moffat and Ashihara 2002). To date, no relationship be-
tween these two cDNAs and the Arabidopsis ATase gene
cloned by Senecoff et al. (1996) has been reported.
Description of other genes involved in the de novo synthe-
sis of purine nucleotides are also available (reviewed by Mof-
fat and Ashihara 2002). A gene encoding for 5-aminoimida-
zole ribonucleotide (AIR) synthetase was described in Arabi-
dopsis by Senecoff and Meagher (1993). This gene had high
homology with several other AIR synthetase genes from bac-
terial origins. Several Arabidopsis clones encoding 5-amino-
imidazole-4-N-succinocarboxymide ribonucleotide (SAICAR)
synthetase were shown by Senecoff et al. (1996). One of
these clones, PUR7, was highly expressed in the leaves and
stems. The involvement of PUR7 during active growth was
demonstrated by Senecoff et al. (1996), who observed in-
tense GUS staining in actively-dividing regions of root, shoot
and flowers of transgenic plants containing a PUR7 : GUS
translational fusion. In the same study, it was also demon-
strated that experimental inhibition of the purine de novo
pathway resulted in increased PUR7 expression. Reports of
Arabidopsis cDNAs or genomic sequences encoding for
many enzymes of the de novo synthesis of purine nucleoti-
des, with the exception of formyl amidine ribonucleotide syn-
thetase and GMP synthetase, are currently available. How-
ever, only a few of them have been characterized (see Moffatt
and Ashihara 2002).
Although progress towards the characterization of genes
encoding for guanine/guanosine and adenine recycling en-
zymes have been made in recent years, the enzymes that sal-
vage adenine and adenosine have been studied to a greater
extent, partially because of their involvement in cytokinin me-
tabolism (see Mok and Mok 2001). In the Arabidopsis gen-
ome there are five sequences encoding for adenine phospho-
ribosyltransferase-like enzymes, designated as APT1– 5. To
date, expression studies of these genes are only available for
APT1– 3. Studies conducted by Moffatt and Somerville (1988)
suggested that APT1 is the most abundant isoform since al-
most no adenine phosphoribosyltransferase activity was de-
tected in APT1 mutant plants. The involvement of this enzyme
during periods of intense growth and development (see
above) was also confirmed by molecular studies. In plants
transformed with a construct expressing GUS from the apt1
promoter, GUS activity was particularly high during cambial
development in both the stem and root (see Moffatt and Ashi-
hara 2002). In agreement with this result, Sterky et al. (1999)
recovered several adenine phosphoribosyltransferase se-
quences from expressed sequenced tags (EST) libraries gen-
erated from cambial tissue of poplar.
In plants, adenosine salvage is mainly mediated by AK ac-
tivity. The Arabidopsis genome encodes two AK proteins
which share high similarity (92 %) at the amino acid level. Ex-
pression of these genes was reported in all organs tested
(see Moffatt and Ashihara 2002). Phenotypic studies of AK-
deficient plants created by sense and antisense silencing
have revealed the involvement of AK in SAM-dependent
transmethylation activities (Weretilnyk et al. 2001).

Relative to the de novo and salvage synthesis of purine nu-
cleotides, there is virtually no information on the purine degra-
dation pathway in plants. To date, in fact, only a few genes
encoding for enzymes of purine catabolism have been char-
acterized (see Moffatt and Ashihara 2002).
4.2 Pyrimidine nucleotide synthesis and degradation
Of the enzymes involved in the de novo pathway of pyrimi-
dine synthesis (see Fig. 2), carbamoyl phosphate synthetase
is composed by two subunits encoded by individual genes in
the Arabidopsis nuclear genome (Williamson et al. 1996).
Amino-acid analysis of these subunits revealed the presence
of a chloroplast transit sequence, consistent with the bio-
chemical evidence for the chloroplast localization of this en-
zyme activity (Newman et al. 1994). Cloned cDNAs from pea
encoding two different isoforms of aspartate transcarbamoy-
lases were reported by Williamson and Slocum (1994). These
two cDNAs also encoded polypeptides with chloroplast trans-
it-peptide sequences.
Conversion of orotate to UMP is carried out by a single
polypeptide that is termed UMP synthase, which has both
OPRT and orotidine 5-monophosphate decarboxylase activity
(discussed earlier). Several cDNAs encoding for UMP syn-
thase have been isolated in tobacco and rice (Oryza sativa)
(see Moffatt and Ashihara 2002). Another well characterized
enzyme is UMP kinase, which phosphorylates UMP to pro-
duce UDP (see Fig. 2). This enzyme also converts CMP to
CDP. The cDNAs encoding UMP kinase of Arabidopsis and
rice have high similarities with the enzymes in yeast (45 %
and 49.7%), and mammals (56 % and 53 %) (Park and Thorn-
burg 1999). Only very fragmentary molecular studies on sal-
vage and degradation of pyrimidine nucleosides are avail-
able, and have been recently summarized by Moffatt and
Ashihara (2002).
5. Conclusion and Perspectives
From the above discussion, it is clear that studies on metabo-
lism of purine and pyrimidine nucleotides in plants have been
limited. This situation is probably largely because the activ-
ities of the constitutive enzymes are low and unstable. Most
metabolic intermediates of the de novo pathway are not com-
mercially available. Also, the presence of a salvage pathway,
in addition to the standard biosynthetic and degradative rou-
tes, renders studies on nucleotide biosynthesis difficult to
carry out. However, the results from biochemical investiga-
tions obtained up to the end of the last century indicated that
the biosynthetic pathways of purines and pyrimidines in
plants were similar to those in other organisms, although or-
ganization of the participating enzymes are different from
other eukaryotes. The de novo pathway of purine and pyrimi-
dine biosynthesis seems to be constitutive, but salvage en-
Plant nucleotide metabolism 1289
zymes may have a special role in the activation of resting
cells and in response to environmental change. Furthermore,
unique expressions of enzymes whose functions are not yet
clear are noted. Thus, for example, adenosine nucleosidase
and nucleoside phosphotransferase, which are almost inac-
tive in seeds and dormant tubers, become quite active during
germination in sprouts. Very limited information is presently
available on molecular aspects of nucleotide metabolism.
Acknowledgements. The research by the authors cited in this article
was supported by research grants from the Ministry of Education,
Science, Sports and Culture of Japan and the Salt Science Founda-
tion (for HA), and the Natural Sciences and Engineering Research
Council of Canada (for TAT).
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