Plant Cell, Tissue and Organ Culture 69: 1–34, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

1

Review

Role of polyamines in the ontogeny of plants and their biotechnological

applications

Harsh Pal Bais1,2 & G.A. Ravishankar1,∗

1 Department of Plant Cell Biotechnology, Central Food Technological Research Institute, Mysore 570 013, India
(2 present address: Department of Horticulture and Landscape Architecture, 205 Shepardson, Colorado State
University, Colorado, USA; ∗ requests for offprints; Fax: +91-821-517-233; E-mail: pcbt@cscftri.ren.nic.in)

Received 22 August 2000; accepted in revised form 25 September 2001

Abstract
Recent developments in the metabolism and function of polyamines in plants is presented. Polyamines appear to
be involved in a wide range of plant processes, however their exact role is not completely understood. In this
review, the metabolic pathways involved in polyamine biosynthesis and degradation are explained, along with
the transport and conjugation of these compounds. The studies involved in the understanding of function(s) of
polyamines using metabolic inhibitors, as well as genetic and molecular approaches are described. Polyamine
metabolism and profound changes in polyamine titres in response to infection by pathogens has been presented. Its
role in adaptation of plants to stress is also presented. Molecular understanding of polyamines and their modulation
in transgenics is also discussed. Further line of work in the understanding of the role of polyamines has also been
focussed.

Abbreviations: PA(s) – polyamines; Put – Putrescine; NCP – N-carbamoylputrescine; Spd – Spermidine;

Spm – Spermine; SAM – S-adenosylmethionine; ACC – 1-aminocyclopropyl carboxylic acid; MTA – 5 -
methylthioadenosine; MTR – 5 -methylthioribose; Dap – 1,3-diaminopropane; Cad – cadaverine; α-DFMA –
α-difluoromethylarginine; α-DFMO – α-difluoromethylornithine; MGBG – methylglyoxal(bis)-guanylhydrazone;
DCHA – dicyclohexylamine; CHA – cyclohexylamine; IAA – indoleacetic acid; GA3 – gibberellic acid; NCP –
N-carbamoyl putrescine; See also abbreviations in Table 1

Introduction pounds are a type of growth regulator or secondary

hormonal messenger (Galston, 1983; Davies, 1987).
Polyamines (PAs) are an important and interest- PAs are found in plant cells at levels significantly
ing group of naturally occurring low molecular higher than those of plant hormones. Their endogen-
weight, polycationic, aliphatic nitrogenous com- ous concentrations required for biological effects are
pounds present in all cells (Galston, 1983). They have in millimolar range. While the question of PAs be-
been implicated in several important cellular processes ing translocated like other growth regulators remains
like cell division, protein synthesis, DNA replication unsettled, there is evidence that PAs are taken up by
and plant response to abiotic stress (Tabor and Tabor, cell suspension cultures (Evans and Malmberg, 1989).
1984; Smith, 1993, 1985; Van Broek et al., 1994). Galston and Kaur-Sawhney (1987) have argued that
They bind to DNA, and are essential for cell viabil- PAs have clear-cut physiological role in plants and
ity (Flink and Pettijohn, 1975). PAs exist in both free therefore should be regarded as members of a more
and bound forms (Evans and Malmberg, 1989). Some loosely defined category of plant growth regulators
authors have postulated that PAs and related com- rather than as hormones per se.
2

Figure 1. Polyamine metabolism and ethylene intervention (for abbreviations and enzyme commission numbers see Table 1).

Polyamine biochemistry, biosynthesis and single multifunctional synthase reportedly carries out
degradation the agmatine aminohydrolase, putrescine transcar-
bamoylase, carbamate kinase, and OTC activities in
As in bacteria, polyamine biosynthesis in plants (Slo- Lathyrus seedings. According to Smith (1965) putres-
cum et al., 1984), is somewhat more complicated, in cine transcarbamylase acting in the reverse direction
that two constitutive pathways lead to Put synthesis. might degrade NCP to Put and carbamoyl phosphate,
Put may be formed directly, through ornithine de- the latter undergoing further hydrolysis to CO2 and
carboxylation by ODC, or indirectly, through a series NH3 .
of intermediates, following arginine decarboxylation In higher plants, the enzyme arginase can also con-
(Figure 1). Arginine decarboxylation, producing ag- vert arginine directly to ornithine, which then can be
matine (Agm), is catalyzed by another pyridoxal utilized in Put synthesis by ODC or metabolized back
phosphate-dependent enzyme, arginine decarboxylase to arginine through the ornithine cycle. The possible
[ADC] (Sindhu and Desai, 1979). Agmatine is then existence of a citrulline decarboxylase, catalyzing the
hydrolysed to N-carbamoyl putrescine (NCP). Beyond pathway leading to Put synthesis has been debated.
this, there is evidence for two apparently unrelated However, the formation of labeled arginine as well as
pathways for the synthesis of Put from NCP with NCP, resulting from the metabolism of [carbamoyl-
14 C]citrulline in Helianthus tissues, suggests that at
the carbamoyl moiety of NCP amidohydrolase, dir-
ectly yielding Put (Yanasigawa and Suzuki, 1982). least some of the NCP may have been derived via the
An alternative mechanism of Put synthesis via a ADC pathway, following citrulline conversion to ar-
multifunctional enzyme, putrescine synthase has also ginine in the ornithine-urea cycle (Kumar et al., 1997)
been proposed (Srivenugopal and Adiga, 1981). This (Figure 2).
 3

Figure 2. Urea–ornithine cycle.

The polyamines, spermidine (Spd) and sper- the MTA molecule are recycled back to methionine
mine (Spm) are synthesized from Put by sub- (Yanasigawa et al., 1981; Kushad et al., 1983).
sequent additions of aminopropyl groups donated In plants, polyamine degradation is carried out
by decarboxylated S-adenosylmethionine (SAM). The by diamine oxidases (DAO) and polyamine oxidases
aminopropyl group additions are catalyzed by spe- (PAO) (Figure 3). Put is oxidized directly to H2 O2 ,
cific aminopropyltransferases, commonly known as NH3 and pyrroline by DAO activity in legumes (Floris
Spd and Spm synthases (Baxter and Coscia, 1973; Hir- et al., 1983). With the possible exception of the
asawa and Suzuki, 1983; Sindhu and Cohen, 1984). DAO characterized from Euphorbia extracts (Rinaldi
It is assumed that two different enzymes, as in other et al., 1982), all of these enzymes exhibit rather
organisms, carry out the Spd and Spm synthase activ- broad substrate specificities, oxidizing Spd and Spm
ities in plants. However, only Spd synthase remains as well as diamine substrates. Specific PAO enzymes
characterized (Sindhu and Cohen, 1984). have been found only in the grasses (Smith, 1983;
Labelling studies have provided evidence that the Smith et al., 1983). These PAO oxidize PAs to 1,3-
aminopropyl moiety incorporated into PAs is derived diaminopropane (DAP), with the release of H2 O2 and
from methionine via SAM (Suresh and Adiga, 1977; pyrroline, or, in the case of Spm oxidation, a substi-
Torget et al., 1979). SAM is decarboxylated by the en- tuted pyrroline (Smith et al., 1983). In the Solanaceae,
zyme SAM decarboxylase (Baxter and Coscia, 1973), where PAs exist largely as cinnamic acid amide con-
and the resulting decarboxylated SAM is utilized in jugates, amine oxidase activity has not been detected
PA biosynthesis. Alternatively, SAM can be metabol- (Flores, 1983). It is interesting to speculate on the
ised successively to 1-aminocyclopropyl-1-carboxylic possibility that free PA titers in these plants may be
acid (ACC) and ethylene, a plant senescence hormone regulated through reversible conjugate formation or
(Adams and Yang, 1979; Liebermann, 1979). Since other types of metabolism, rather than through ox-
PAs and ethylene are both derived from and prob- idative catabolism. Pyrroline is further metabolised
ably compete for a common precursor, i.e., SAM to succinate via a γ -aminobutyric acid intermediate
(Even-Chen et al., 1982; Roberts et al., 1984), from (Terano and Suzuki, 1978), while diaminopropane is
a functional point of view, SAM plays a role in degraded to α-alanine (Flores and Filner, 1984).
the regulation of senescence. 5 -Methylthioadenosine It should be noted that we have limited our dis-
(MTA), a common metabolic byproduct of both ACC cussion here to the synthesis of the major PAs found
synthase and aminopropyltrasferase activities, is de- in most plant tissues, namely, Put, Spd, and Spm.
graded to 5 -methylthioribose (MTR) by the enzyme The synthesis of cadaverine (Cad), formed as a res-
MTA nucleosidase (Kushad et al., 1982), the first ult of lysine decarboxylation (Schoofs et al., 1983),
step in a pathway through which various moieties of is known in some plants, while numerous other PAs
4

Figure 3. Polyamine oxidation and degradation (modified from Evans & Malmberg, 1989) (for abbreviations and enzyme commission numbers
see Table 1).

have been reported in algae (Hamana and Matsuzaki, trol of Put synthesis during plant development, or
1982) and in higher plants (Smith, 1981). The most in response to stress or hormonal induction of de-
common PAs are diamines putrescine (Put), cadaver- carboxylase activity, as is discussed in the following
ine (Cad), triamine spermidine (Spd) and tetramine sections. Put incorporation into PAs can be blocked by
spermine (Spm). In general, prokaryotic cells contain methylglyoxal(bis)guanylhydrazone [MGBG] (Pegg,
fairly large amounts putrescine, small quantities of 1983), an inhibitor of SAM decarboxylase, while
spermidine and no spermine, while eukaryotes have spermidine synthase is inhibited by dicyclohexylam-
little Put, more Spd and considerable Spm. In addition monium sulfate [DCHA] (Sindhu and Cohen, 1984).
to the usual PAs, some unusual PAs such as caldine, The extracellular PAO can be inhibited by carboxyl
thermine and calcopentane, have been reported in a reagents, such as α-hydroxyethylhydrazine (Kaur-
variety of thermophiles (Oshima et al., 1988; Bagga et Sawhney et al., 1981).
al., 1997). It has been shown by Lan et al. (2000) that ODC
overexpression in the skin of TG.AC v-Ha-ras trans-
genic mice induces the formation of spontaneous skin
Inhibitors of polyamine metabolism and carcinomas. Treatment of ODC Ras double transgenic
biosynthesis mice with α-DFMO causes a rapid regression of these
spontaneous tumors. DFMO induced apoptosis in HC
11 mouse mammary epithelial cells in a dose- and
Early studies on the role of PAs in various growth
time-dependent manner. Apoptosis induced by ODC
and development processes were largely correlative,
since in vitro polyamine titers could not be eas- inhibition was associated with a rapid increase in
ROS concentration in HC 11 cells observed within
ily manipulated. Irreversible inhibitors of the en-
1 h after DFMO treatment. The administration of 50
zymes of polyamine metabolism have been developed
µM of Put lowered the number of early-apoptotic,
(Stevans and Stevans, 1980; Heby, 1981), allowing
late-apoptotic and necrotic cells. This suggests that
one to examine polyamine biosynthesis and titers and
the disturbance of cellular PA homeostasis by inhib-
a particular growth or developmental response. For
ition of their synthesis enhances mammary epithelial
example, α-difluoromethylarginine [DFMA] and α-
cell susceptibility to apoptosis (Ploszaj et al., 2000).
difluoromethylornithine [DFMO] (Kallio et al., 1981),
irreversible inhibitors of ADC and ODC, (Table 1) ODC and PAs play an essential role in brain cell rep-
lication and differentiation. Work by Slotkin et al.
respectively, have been shown to specifically in-
(2000) indicates that ODC/PA pathway plays a role
hibit these decarboxylases in a number of plants.
in the development of cell signaling, and hence in
The ability to carry out the selective inhibition of
neurotransmission, above and beyond its role in cell
ADC or ODC with these inhibitors has provided us
replication and differentiation in rats.
with valuable insights regarding the metabolic con-
 5
Table 1. Abbreviations of enzymes and their enzyme commission numbers

Enzyme Abbreviation Enzyme commission number

Arginine decarboxylase ADC E.C.4.1.1.19

Ornithine decarboxylase ODC E.C.1.1.17
Diamine oxidase DAO E.C.1.4.3.6
Polyamine oxidase PAO E.C.1.5.3.11
1-Aminopropyl carboxylic acid synthase ACC Syn E.C.4.4.1.14
Spermidine synthase Spd syn E.C.2.5.1.16
Spermine synthase Spm syn E.C.2.5.1.22
Ornithine carbamoyl transferase OCT E.C.2.1.3.3
Putrescine-aminopropyl transferase Put APT E.C.2.5.1.16
S-Adenosylmethionine decarboxylase SAMdc E.C.4.1.1.50
Ornithine transcarbamoylase OTC E.C.2.5.4.19
1-Aminopropyl carboxylic acid oxidase ACC oxidase E.C.2.3.3.21
Arginosuccinate lyase – E.C.4.3.2.1
Arginase – E.C.3.5.3.1
Arginosuccinate synthase – E.C.6.3.4.5
Carbamoyl phosphate synthase – E.C.6.34.1.6
γ -Aminobutylaldehyde dehydrogenase – E.C.1.2.1.19

Since the availability of specific and potent in- growth of wheat plants for the duration of the 28-day
hibitors of PA metabolism, rapid strides have been observation period.
made to understand the close relationships between In spindle tree (Euonymus europaeus L.), DFMO
PAs and the physiological processes as well as of bio- rapidly inhibited Put accumulation and growth in
chemical mechanisms at the molecular level in many non-embryogenic calli and highly stimulated rooting
systems (Evans and Malmberg, 1989; Balasundaram activity. DFMO partially inhibited Put accumulation
and Tyagi, 1991). Torrigiani et al. (1987) have re- in embryogenic calli. This inhibition had no effect
ported the effect of dicyclohexylamine (DCHA) on on callus growth but significantly reduced the time
endogenous polyamine titers during cell cycle of Heli- of emergence of roots and highly stimulated somatic
anthus tuberosus explants. In addition, chemothera- embryo production (Bonneau et al., 1995).
peutic implications of PA biosynthesis inhibition were Canaline (an ornithine analogue) and canavanine
also realized (Galston and Weinstein, 1988; Pegg, (an arginine analogue) inhibited root and shoot form-
1988; Walters, 1989). ation in in vitro hypocotyl segments of Euphorbia
Polyamine inhibitors have been used for control of esula. This inhibition of root formation by canavanine
growth and differentiation in several systems, which was partially reversed by arginine. Ornithine, a pre-
are of application value. cursor of Put via the ODC pathway also inhibited the
Bean plants protected from infection by DFMO ap- root formation (Davis, 1997).
plication did not exhibit any morphological alterations DFMO and DFMA inhibited the root and shoot
or reduction in growth rate as compared to unsprayed, formation in E. esula. The inhibition of shoot form-
uninfected controls. By contrast, unprotected, infected ation was not reversed by exogenous addition of Put
plants showed a marked reduction in height (Rajam et but the root formation was partially restored by Put.
al., 1985). Walters (1989) observed that DFMO at 0.4 IAA reduced the inhibitory effects of DFMO+DFMA
mM had no effect on either growth or total intracellu- on both roots and shoots (Davis and Olson, 1994).
lar PA concentrations in Vicia faba plants. Weinstein The addition to the culture medium of exogenous
et al. (1987) also observed that PA inhibitors had no PAs caused a stimulation of the growth of aerial parts
apparent effects on the growth of wheat plants. Fur- of potato cuttings. Spm stimulated the tuber develop-
ther studies by Mussell et al. (1987) showed that even ment, Spd inhibited it and Put had no effect. DFMO
at 20 mM, DFMO apparently had no effects on the and MGBG had opposite effects to that of Put and Spd
6

and the inhibitor effects were partially reversed by PAs species. Their general conclusions were that although
(Feray et al., 1993). there was an increase in the polyamine contents of
DFMA and DFMO were found to protect soybean cotyledons and endosperms during germination, the
seeds against infection by Colletotrichum truncatum. increase was not frequent in those organs exhibiting
The inhibition of fungal growth was reversed by the rapid cell division. Mader and Hanke (1997) ob-
addition of Put or Spd (Gamarink et al., 1994). served that polyamine sparing might be involved in
In maize callus, short-term treatments at high the prolongation of cell division due to inhibition of
doses of DFMA significantly increased the number of phenylpropanoid synthesis in cytokinin-starved soy-
regenerated buds as against the control. By contrast, bean cells. The report by Berta et al. (1997) showed
long-term treatment at low doses reduced the number the absolute requirement of PAs for cell wall elong-
of plantlets (Guergue et al., 1997). ation and modification that explains the cell wall hy-
pertrophy was related to inhibition of PA biosynthesis.
This, in turn may suggest the involvement of PAs in
Polyamines in plant growth and development interaction of cell wall components that aids cell wall
rigidity and cell-to-cell adhesion.
Cell division Recent reports by Sawka et al. (1998) showed the
involvement of PAs in signal transduction with special
Several studies have shown correlations between in- reference to Ca2+ ions. They supported the hypo-
crease in polyamine levels with cell division and a thesis of transportation of Spd/Spm within protoplasts
drop in polyamines during reduction of metabolic through a carrier-mediated mechanism (Antognoni et
activity. Heimer et al. (1979), for example, demon- al., 1994; Sawka et al., 1997). Sawka et al. (1998)
strated a significant level of ODC activity, correlated found that Spd/Spm may result in change in distribu-
with cell division frequency in tobacco suspension cul- tion of Ca2+ ions, whereas Put did not function that
tures and tomato ovaries. Berlin and Forche (1981) effectively, due to presence of two positively charged
reported that low doses of DFMO (an ODC inhibitor) amine groups, which further lead to the accumulation
inhibited cell division in tobacco cell cultures, while of free Ca2+ to enhance transduction. It is reason-
cell enlargement continued. The observed cell enlarge- able to conclude that Ca2+ ions may be involved in
ment without division suggests that DFMO blocks G- mechanism of PA action in plant cells (Bush, 1995),
phase in the cell cycle. However, it remains to be elu- which may interest researchers to facilitate the work
cidated. Walker et al. (1985) observed not only a rapid on cytosolic content of Ca2+ ions.
accumulation of polyamines correlated with rapid cell
division in Acer saccharum seedlings, but also an Embryogenesis
inhibition of cell division and a drop in polyamine
titers with addition of D-arginine, D-arginine+DFMO, Several studies have investigated the role of polyam-
MGBG (a SAMdc inhibitor), or CHA (a spermidine ines and particularly ADC in carrot somatic embryo-
synthase inhibitor). Cell elongation was unaffected. genesis. Montague et al. (1979) were the first to
In studies on maize roots Schwartz et al. (1986), demonstrate that there was a significant rise in ADC
found a high Spm content in primary root apices activity and in Put pools when carrot cultures were
and in decapitated roots as laterals formed, using la- shifted from callus medium to embryogenesis me-
belled DFMO and thymidine. ODC was shown to be dium. Feirer et al. (1984) found that the inhibitor
localized primarily in the meristematic zones. DFMA (an ADC inhibitor) blocks the transition from
In oat protoplasts, application of the polyamines disorganized growth into somatic embryogenesis, and
Cad, Spd and Spm stimulated both DNA synthesis that the addition of Put with DFMA restored the em-
and a limited amount of mitotic activity (Galston et bryogenic potential. However, DFMA did not block
al., 1978). It has been shown that during exogenous growth of the carrot cells on callusing medium. So
applications of Arg (1–10 mM) or Put, Spd or Spm the Put requirement seems to be unique to the trans-
to medium, Put was most effective, although no clear ition to embryogenic growth. Fienberg et al. (1984)
dose dependency was observed for any of the amines examined polyamine synthesis in a carrot cell mutant
(Wu and Kuniyuki, 1985). that would not undergo embryogenesis. The cell line
Felix and Harr (1985) surveyed the PA contents W001 C had high internal levels of auxin that presum-
in different organs of seedlings of a number of plant ably inhibit this transition. When placed in medium
 7

without auxin, the mutant line also failed to show the Roots
typical increase in polyamine content and in ADC and
SAMdc activities that wild-type cells display. The possible role of polyamines in root formation
Garrido et al. (1995) observed an increase in both and growth has been investigated in several plant sys-
Put and Spd bound to macromolecules during embryo- tems, mostly in Phaseolus and in Vigna, In Phaseolus,
genic induction from isolated mid-binucleate pollen of Jarvis et al. (1985) found evidence that PAs are not
Nicotiana tabacum. During embryogenesis a gradual only correlated with root initiation and early growth,
disappearance of ODC was observed, however ADC but may be essential for these process. Treatment
activity decreased along the induction period and in- of cuttings with indole-3-butyric acid increased the
creased again during the embryo formation (Minocha levels of Put, Spd and Spm in hypocotyls, prior to
et al., 1993). In yet another study using hypocotyl increase in root number. Application of the SAMdc
segments of Solanum melongena an increase in the inhibitor, MGBG reduced endogenous levels of Spm
content of Put at fully matured stage has been recor- and Spd, raised Put levels, inhibited the indolebutyric
ded (Sharma and Rajam, 1995; Yadav and Rajam, acid induced rooting. Palavan-Unsal (1987) determ-
1997). Similarly in Camellia conjugated Put and sol- ined through the use of cadaverine that ADC was the
uble conjugated Spd increased during the formation major branch of Put synthesis involved in root growth
and development of globular embryo (Pedroso et al., in Phaseolus, although both ODC and ADC activities
1997). CHA and mgBG reduced embryogenesis and were high in the root apex. Inhibition of ADC re-
Spd content and increased Put but not Spm (Khan and duced PA titers and growth, both of which could be
Minocha, 1991). However MGBG did not show any partially reversed upon addition of Put. Applications
inhibitory effects on somatic embryo formation in al- of α-methylornithine slightly reduced root length and
falfa (Meijer and Simmonds, 1988) and Picea glauca inhibited ODC activity. Kakkar and Rai (1987) cor-
(Kong et al., 1998). roborated earlier findings that Spm is associated with
A significant genotype difference in polyamine enhanced rooting and found that Spd in combination
requirement has been noted in a study of somatic with indoleacetic acid increased carbohydrate content.
embryogenesis in two alfalfa lines (Meijer and Sim- It may be significant that the above studies have found
monds, 1988). Both embryogenic genotypes showed Spm effective in enhancing rooting correlated by Du-
Put accumulation during induction on medium with mortier et al. (1983) finding Spm only in the apical
auxin and both exhibited sharp decreases in Put con- region of maize roots.
centration upon transfer to the differentiation me- Friedman et al. (1985) failed to find any stimula-
dium without auxin. The polyamine inhibitors CHA, tion of rooting in Vigna hypocotyls, but did record
MGBG, DFMO, and DFMA all reduced polyamine an increase in PAs after indolebutyric acid-induced
contents in both genotypes. rooting. Additional studies with [14 C]ornithine and
A few reports indicate that the PAs and ethyl- [14 C]arginine by the same authors implied that both
ene play an important role in regulation of somatic ethylene and polyamine pathways contributed to the
embryogenesis of cultured cell/tissues (Biddington, IBA-stimulated increase of polyamines.
1992). The recent report by Patil et al. (1999) shows Working with apple, Wang and Faust (1986) noted
that ethylene alone does not aid in somatic embryo- a considerable increase in polyamine levels accom-
genesis. They inferred that Put plays the key role in panying the induction and growth of roots. But, in
regulating both PA and ethylene biosynthesis, which contrast to the reports on Phaseolus, use of the in-
further leads to increased somatic embryogenesis. hibitor DFMO was more effective than DFMA in
However, the involvement of other endogenous growth reducing fresh weight increase, indicating a major role
regulators, gases other than ethylene in influencing for ODC rather than ADC. Tiburcio et al. (1987),
somatic embryogenesis cannot be ruled out. These res- observing root organogenesis in tobacco callus cul-
ults imply that the requirement for polyamines in initi- tures, found root production to be inversely related
ation of somatic embryogenesis may not be universal to Put and alkaloid titers. The addition of DFMA or
and is very much system specific. D -arginine decreased the Put and alkaloid titers and
promoted rooting, implying the involvement of the
ADC branch of polyamine synthesis in production of
Put derived alkaloids and an influence of PA over in
vitro organogenesis. Root differentiation in tobacco
8

was found to depend on both a rise in Put titres and they would not. Although the appearance of the con-
a drop of pH, each of these two factors acting inde- jugates occurred late in development at both temperat-
pendently. Inhibition of Put biosynthesis was found ures, there clearly was no correlation with flowering
to prevent root initiation while exogenous Put sup- per se. However based on topping and leaf growth
ply reversed this effect and thus Put could be used analysis, Cabanne et al. (1981) proposed that the con-
as a marker for root differentiation (Tiburcio et al., jugates might be related to the appearance of ripening
1989). Chriqui et al. (1986) have shown that there to flower. Presumably the plants at 30◦ C were ripened
exists a synergistic effect between auxins and ornith- to flower but were repressed from expressing it.
ine in rhizogenesis in Datura innoxia leaf explants. Dumas et al. (1981) have examined the levels of
Although addition of PAs to auxin-containing me- polyamine-conjugates in shoot apices of the Nicotiana
dium did not enhance rhizogenesis, exogenous Put stock RMB7, which is not capable of flowering. This
may have promoted growth of the roots (Chriqui et al., hybrid plant, which arose from a program of inter-
1986). In the extension of the work done by Chriqui et specific crossing of Nicotiana rustica with Nicotiana
al. (1986), Baraldi et al. (1995) showed the synergistic tabacum cv. Xanthi, showed reduced conjugate levels.
activity of auxins and PAs in relation to the differential However, in this interspecific hybrid, it is very dif-
in vitro root induction in two pear cultivars. ficult to decide what the proper control comparison
In cultured roots of Hyoscyamus albus, putrescine- is, because RMB7 is not isogenic with any cultivar.
N-methyl transferase was suggested to function in Changes in polyamine or polyamine-conjugate levels
vivo, as the first committed enzyme, which interme- might be the result of a genetic factor that is independ-
diates the tropane alkaloid biosynthesis and nicotine ent of the flowering effect.
biosynthesis (Hibi et al., 1992). Hayyim et al. (1994) Because of the difficulties in conducting appropri-
showed that addition of α-difluromethylornithine ate experiments with intact plants, several investig-
(DFMO), resulted in enhancement of growth of hairy ators have turned to analyses of floral initiation and
root cultures of tobacco, whereas polyamine levels development in organogenic cultures and in vitro sys-
were lower. However, Altabella et al. (1995) have tems. The tobacco thin-layer method of Tran Thanh
shown enhanced growth under the rol A or rol A/rol Van (1973, 1981) has been used to study the effects
B/rol C, expression. However, rol A resulted in en- of polyamines and their inhibitors on floral initiation
hanced polyamine levels whereas rol B and rol C in- in vitro. Torrigiani et al. (1987a) measured free and
hibited polyamine accumulation in hairy root cultures bound polyamines at different times during the cul-
of tobacco. ture of thin layers taken from floral-determined tissues
Work done in our laboratory with the transformed and from vegetative stem tissues. The rates of ap-
root cultures has shown that PAs, especially Put, play pearance of Put, Spd and Spm were slightly different
an important role in root growth and differentiation. In between the vegetative and floral bud-forming tissues.
hairy root cultures of Cichorium intybus, Put influence But both showed significant increase (10–20-fold) first
was confirmed by the use of PA biosynthetic inhibit- of Spd, then of Put levels. The polyamine-conjugates,
ors, while further addition of Put in inhibitor-treated both soluble and insoluble, were found at roughly 10-
samples resulted in restoration of growth (Bais et al., fold higher levels than the free PAs, and the levels of
1999a, 1999b). Similar results were obtained while the Put-conjugates increased significantly during the
working with hairy root cultures of Beta vulgaris and course of explant development. These results show a
Tagetes patula wherein PAs generally influenced root correlation of PAs and conjugates with bud formation,
growth and differentiation (Bais et al., 2000a). but no differences between floral and vegetative pro-
grams. These results imply that PAs may play a role
Floral initiation in bud formation in general, as opposed to floral bud
formation in particular.
Cabanne et al. (1981) have studied the accu- Kaur-Sawhney et al. (1988) have reported more
mulation of various hydroxycinnamic acid amides dramatic results in switching to development pro-
(or polyamine-conjugates, primarily mono- and di- grams. They used two basic media for culturing floral
caffeoylputrescine) in Nicotiana tabacum cv. Xanthi stem epidermal peels. One led to development of floral
plants. They found accumulation of the conjugates buds in ‘floral medium’, while the other to develop-
in apical shoots and leaves of plants grown at 20◦ C, ment of vegetative buds in ‘vegetative medium’. The
where they would flower, as well as at 30◦ C, where underlying media difference was a 10-fold higher level
 9

of cytokinin in the ‘vegetative medium’. Spd levels flowering for a short time, as opposed to permitting
were 4.5-fold higher in explants cultured on floral bud a change of the tissue to a true vegetative mode of
medium than in those cultured on vegetative bud me- growth.
dium. These results led to further testing of the effects The work done in our laboratory has shown that
of exogenous Spd on the buds formed on the vegetat- exogenous feeding of Put and silver nitrate (AgNO3 ),
ive medium and the effects of cyclohexylamine (CHA) a known inhibitor of ethylene action (Yang and Hoff-
on the buds formed on the floral medium. Spd in the man, 1984), influenced the morphogenesis in chicory
range 0.5-5 mM decreased the number of vegetative shoot cultures (Bais et al., 2000b). Put and AgNO3
buds on vegetative medium by 70% and caused the ap- influenced morphogenesis with respect to shoot mul-
pearance of a number of floral buds that had not been tiplication and in vitro flowering. Under these treat-
seen previously on the vegetative medium. CHA at ments the shoot apices flowered without vernaliza-
10–20 mM caused both 75% reduction in the amount tion, otherwise field grown plants required vernal-
of Spd in the explants and a significant shift from floral ization (Demeulemeester and DeProft, 1999). This
buds to vegetative buds, on what was otherwise the was also evident from the polyamine inhibitor stud-
floral medium. The simultaneous addition of 10 mM ies carried out with the chicory shoot explants (Bais
CHA and 1 mM Spd provided a partial reversal of the et al., 2000b). We found that the morphogenesis in
CHA-induced shift, suggesting that the depletion of chicory shoots, flowering and shoot multiplication
Spd really was an important underlying cause of the were governed by the interplay between polyamine
shift. and ethylene biosynthesis (Bais et al., 2000b). This
The CHA-Spd induced shoots observed by Kaur- was evident by the lower ethylene production in Put-
Sawhney et al. (1988) could be interpreted as Spd treated samples, possibly due to the over-utilization of
masking the effects of the high cytokinin in the ve- S-adenosylmethionine by Put and hence the morpho-
getative medium, or it could imply that Spd had some genetic response (Bais et al., 2000b).
completely different role in promoting floral initiation.
The levels of auxin and cytokinin that affected the Floral development
floral-vegetative shift in this system were 1–10 µM,
whereas the levels of PAs and polyamine inhibitors Initial reports from Heimer and Mizrahi (1985)
with similar effects were 1–20 mM. This difference demonstrated high ODC levels specifically in the de-
in required exogenous concentration could either be veloping ovaries of the tomato flower. Subsequently,
related to effects such as poor polyamine transport they have demonstrated that feeding tomato ovaries
(Young and Galston, 1983a) or large initial pools of with the inhibitor DFMO will block their develop-
PAs; or could indicate that the PAs are less likely to be ment, whereas DFMO plus Put allows the ovaries to
natural regulators than the cytokinins. grow normally (Heimer and Mizrahi, 1982). Slocum
A further complication is that a number of differ- and Galston (1985a) have characterized the enzyme
ent variables have now been reported that cause shifts activities, polyamines, soluble polyamine-conjugates,
in the organogenic program, including pH, cytokinin and insoluble polyamine-conjugates in developing to-
amount and type, oligosaccharins (Tran Thanh Van, bacco ovaries. ODC-specific activity rose about 3-fold
1981), as well as polyamines. One has the sense that during the course of ovary development and fruit set;
either the system is so plastic that a variety of sig- this increase was correlated with a doubling in the free
nals can induce a change, or that the real underlying Put titer but with no significant change in the Spd
variable has not yet been found. The implication is and Spm titers. At its peak, the ODC activity was
either that in the intact plant a variety of signals can 140-fold that of ADC. More than 90% of the total
be alternatively associated with the floral/vegetative content for all three PAs was found in the bound con-
switch, or that the in vitro and in vivo system are not jugated form, probably as caffeoyl derivatives. This
always consistent in how a vegetative shoot is defined. result re-emphasizes the need to consider the interplay
Since any shoot will flower eventually, the operational of the free and bound forms of the polyamines. Sub-
difference between a regenerated floral bud and veget- sequently, Slocum and Galston (1985b) demonstrated
ative shoot may be only a very small number of nodes. that DFMO would interfere with tobacco ovary de-
Without a clear definition of a vegetative shoot, based velopment as well. An apparent effect of DFMA on
on a significant developmental assay, it may be that the ovary development resulted from cellular conversion
various ‘vegetative’ media are actually only delaying of DFMA to DFMO by arginase, after which it could
10

inhibit the ODC. A requirement for ADC, not ODC, analyzed genetically, on the basis of a very small num-
has been suggested in the development of avocado ber of seeds (4–16 seeds total per cross) obtained in
fruits (Winer and Abelbaum, 1986). crosses with wild type pollen. Both of these apparently
Martin-Tanguy et al. (1982) have examined the were nuclear dominant mutations with the floral phen-
polyamine-conjugates (hydroxycinnamic acid amides) otypes co-segregating with the polyamine phenotype
in cytoplasmic male sterile lines of Zea mays. These (Malmberg, 1980). The extent to which somaclonal
compounds were found to be absent in anthers from variation, cell culture artifacts, and multiple mutations
plants with the Texas male sterile cytoplasm, and arising from mutagenesis might have contributed to
present in plants that also contained the appropriate the floral morphology aberrations is unclear.
nuclear restorer gene. Analysis of the post-fertilization In Sinapis alba, the titres of free and conjugated
events of cob and grain development and maturation Put increased early and markedly in leaf exudates dur-
showed no changes in the hydrocinnamic acid content ing floral transition, coinciding with movement of the
in male sterile and fertile lines. These changes were floral stimulus out of the induced leaf. Spraying with
developmentally restricted to the affected anthers. DFMO decreased the flowering response of induced
Gerats et al. (1988) began a study with Petunia plants. This effect was substantially reversed by a
mutants that had been identified as having altera- simultaneous application of Put to the roots. It was
tions in floral morphology. Polyamine, polyamine- concluded that the extra Put synthesized in induced
conjugate, and enzyme activities were measured to see leaves is a necessary component of the floral stimulus
if any changes could be found. A polymorphism was in S. alba (Havelange et al., 1996).
observed in comparing two wild-type lines of Petunia, Working in our laboratory with the shoot cultures
with high and low Put levels segregating as a simply of both normal and transgenic chicory we have found
inherited trait. This result implied that not all changes that endogenous polyamine content decreases with the
in polyamine levels or ratios have to be associated floral development and is maximum during anthesis.
with floral morphology changes. However, one out of However their level reduced in seeds obtained in vitro
the four floral morphology mutations screened showed (Bais, 2000).
significantly higher Put levels and ADC in flower Although each of the studies discussed above has
and older levels, but no younger leaves (Gerats et al. its limitations, the collective evidence indicates that
1988). The Put elevation co-segregated with the floral PAs (or their conjugates) significantly affect floral ini-
morphology change in both of the wild-type genetic tiation and floral development. PAs may be required
backgrounds. The phenotype of the lesion, alfalfa, is during crucial differentiation steps, or they may be
quite chaotic, with floral parts turning into other floral part of the hormonal regulation of sex development in
parts on a frequent and irregular basis; the vegetative plants. An alternative explanation is that similar in-
part formed later in development are also abnormal, terplay of many other metabolic pathways would also
with smaller leaves and prolific branching (Goren et produce systematic floral initiations.
al., 1982a). This result implies that screening plants
selected for their unusual floral phenotype may find
some significant changes in polyamine synthesis. Pollen
Work of Malmberg (1980) and Hiatt and Malmberg
(1988) showed the use of tobacco cell cultures to select The possible role of polyamines in pollen develop-
variants resistant to two inhibitors of polyamine syn- ment has been given very limited attention. Using
thesis, MGBG and DFMO. Plants were regenerated radioactively labeled arginine, Bagni et al. (1981)
from 14 of these lines, while a number of lines have demonstrated that synthesis of polyamines precedes
regenerated only into green coral-like calli. Among the emergence of apple pollen tubes. Speranza et al.
the 14 regenerated lines, two were extreme dwarfs (1983) found little effect of exogenous polyamines on
and did not flower (ts4 and Dfrl); each of these two apple pollen germination, although under Ca2+ de-
lines had multiple pleiotropic effects on the polyam- ficiency exogenous spermine did have a promotive
ine pathway including low levels of ODC. The cell effect. Prakash et al. (1988) studying in vitro pollen
culture-derived polyamine variants upon regeneration tube growth in Catharanthus demonstrated a promot-
showed aberrations in floral development. Generally ive effect of 0.01 mM Spd on pollen germination.
these aberrations were so severe that the plants were The presence of MGBG at 0.5 mM, without Spd ad-
sterile. Two lines, Mgr 3 and Mgr 12 were partially dition, reduced germination percentage, and 15 mM
 11

totally inhibited germination. Apparently no attempt Spd (Nathan et al., 1984). Correlations have also been
was made to apply MGBG and Spd simultaneously. reported between rates of cell division and Put and Spd
levels in avocado pulp (Apelbaum, 1986) and between
Fruit development onset and rapid cell division and ODC activities in
post-fertilization tobacco ovary tissues (Slocum and
Several lines of evidence have implicated a role Galston, 1985a).
for polyamines in fruit development (Evans and Costa and Bagni (1983) showed in apples that
Malmberg, 1989; Egea-Cortines and Mizrahi, 1991). spraying PAs at millimolar concentrations on flowers 9
Evidence comes from: days after full bloom increased both fruit set and yield,
– experiments of exogenous application of PAs dur- apparently by increasing fruit growth rate during the
ing fruit set or initial steps of fruit development, stage of rapid cell division. Flower bud formation was
– changes in their endogenous levels during that also increased. Cohen et al. (1982) have shown the
process, involvement of ODC in early stages of tomato fruit
– changes in the activity of the enzymes involved in development this was further confirmed by the use of
PA biosynthesis, and two ODC inhibitors α-DFMO and α-methyl ornithine,
– experiments with specific inhibitors of biosyn- wherein inhibition of fruit development was observed.
thetic enzymes. A study of exogenous Put applications on apple has
During initial steps of fruit development, changes in reported increased fruit set with one cultivar on one
the activity of ODC and/or ADC, enzymes involved rootstock but no effect on another rootstock or with
in Put biosynthesis, have been found in tomato (Ly- two other cultivars (Volz and Knight, 1986). Increased
copersicon esculentum) (Heimer et al., 1979; Cohen et fruit set has also been reported in olives following ap-
al., 1982; Teitel et al., 1985), Phaseolus vulgaris (Pa- plication of Put at high concentration during flowering
lavan and Galston, 1982), mandarin Citrus reticulata (Rugini and Menucuccini, 1985).
(Nathan et al., 1984), tobacco (Nicotiana tabacum) Fruit set in tomato can be induced artificially by
(Slocum and Galston, 1985a), avocado (Persea amer- treatment of unpollinated ovaries with growth regu-
icana) (Apelbaum, 1986; Kushad et al., 1988) and lators. A transient increase in both ODC and ADC
apple (Malus domestica) (Biasi et al., 1991). In tomato activities was shown after treatment with GA3 and 2,4-
and tobacco, a correlation between increases in ODC D , the latter inducing more effective and rapid changes
activity and cell division (or active cell growth) have (Alabadi et al., 1996).
been observed, suggesting that ODC is the primary In tomato, there was a transient increase of the
enzyme in the regulation of Put biosynthesis during ODC mRNA levels, which showed a maximum at 8
the initial fruit growth. In general, it has been sug- days post anthesis. Unpollinated ovaries of partheno-
gested that changes in ODC may regulate cell division carpic fruits at 1 day post anthesis showed relatively
in actively growing tissues, whereas ADC may regu- high levels of ODC mRNA. However, treatments with
late cell extension and secondary metabolic processes 2,4-D or GA3 induced a transient increase of the ODC
such as alkaloid biosynthesis (Tiburcio et al., 1990). mRNA levels with a maximum in the expression at 5
In unpollinated pea (Pisum sativum L.) ovaries, the days post anthesis for 2,4-D treated ovaries and at 8
total amount of endogenous PAs increased after GA3 days post anthesis for GA3 treated ovaries (Alabadi
treatment, but remained nearly constant or decreased and Carbonell, 1998).
slightly in untreated ovaries (Carbonell and Navarro,
1989). It has been shown that there is an increase in Seeds
the total amount of Put, Spd and Spm in pea ovaries
after induction of fruit development with plant growth The ODC activity in germinating barley seeds can be
regulators (Carbonell and Navarro, 1989) and a high induced by GA3 and IAA. A macromolecular inhibitor
OTC/carbamoyl phosphate synthetase ratio, indicative of ODC exists in barley seeds, which is induced by the
of active Arg synthesis (Garcia-Espana et al., 1989). addition of PAs during germination. The induction of
In apple, Biasi et al. (1988) observed high free ODC by GA3 is not abolished by the addition of PAs,
and bound polyamine levels during the early periods while ODC, which is already inhibited by PAs, can be
of fruit growth, especially bound Spm. In develop- enhanced to control level by GA3 (Kyriakidis, 1983).
ing ‘Murcott’ manderin fruit, correlations were found Dai et al. (1982) have shown that spraying GA3 on 9-
between growth rate and levels of ADC, ODC, Put and day-old light-grown dwarf peas can increase the activ-
12

ity of ADC. In Cicer arietinum the embryonic axis cumulation and adjusting the Put to Spd ratio using
attached to the cotyledons contains a larger amount DFMA or Spd. These observations support the view
of Put, Spd, Spm and Cad than the cotyledons them- that adequate PA levels and their ratios may be im-
selves and larger amount of Put, Spd and Spm than portant for optimum morphogenesis (Bajaj and Rajam,
embryonic axis excised from the whole seeds before 1995, 1996).
germination. Embryotomized cotyledons, on the other DFMO, which inhibits ODC-derived Put synthesis
hand, show far greater polyamine content than whole (Metcalf et al., 1978) was ineffective in reducing the
seeds, implying that the cotyledons are at least a partial PA accumulation and improving the plant regeneration
source of PAs. Nonetheless, Cad is greater in excised in rice (Bajaj and Rajam, 1996). This result supports
embryonic axes, suggesting some control over its syn- the idea that PA accumulation is due to the activation
thesis in this organ by the cotyledons (Gallardo et al., of ADC activity, since it is the predominant pathway
1992). Shiozaki et al. (2000) have reported higher for PA biosynthesis in higher plants (Birecka et al.,
titers of the total endogenous PAs in the seeds of grape 1985; Rajam, 1993; Minocha and Minocha, 1995).
berry. The inhibition of plant regeneration in fresh rice cul-
tures by DFMA particularly at higher concentrations
Influence on de novo morphogenesis (100 µM and 1 mM), may be due to the reduced
levels of cellular PA contents below the threshold
Brassica campestris produced high levels of ethylene needed for regeneration (Robie and Minocha, 1989;
in culture causing abnormal growth and development Helleboid et al., 1995, Minocha and Minocha, 1995).
of the plant (Palmer, 1992), and also inhibiting de DFMA has also been shown to stimulate organogen-
novo shoot regeneration in vitro (Chi et al., 1991; esis in tobacco, which initially had higher Put levels
Pua, 1993). Application of aminoethoxyvinylglycine (Tiburcio et al., 1987) and embryogenic potential in
(AVG) and AgNO3 , which are the inhibitors of ethyl- maize (Tiburcio et al., 1991; Torne et al., 1994).
ene production and action, respectively (Beyer, 1976; The changes in the profile of PAs in Solanum
Yang and Hoffman, 1984), have resulted in high tuberosum were consistently associated with effects
frequency shoot regeneration (70–90%) from cul- on tuberisation. DFMO totally prevented, but DFMA
tured explants of several recalcitrant genotypes of B. only decreased tuberisation. Exogenous Put, in gen-
campestris (Chi and Pua, 1989; Chi et al., 1990, 1991; eral was found to improve growth of leafy shoots,
Palmer, 1992) and B. juncea (Pua and Chi 1993). The rooting and loss of tuber dormancy. The distribution
similar promotive effect of ethylene inhibitors on de of endogenous PAs following treatments that perturb
novo shoot regeneration for other plant species includ- tuberisation support the conclusion that a specific bal-
ing monocots, e.g. Triticum aestivum (Purnhauser et ance of free and conjugated PAs is required for optimal
al., 1987) and Zea mays (Vain et al., 1989; Songstad et progress through tuberisation (Mader, 1997).
al., 1991), has also been demonstrated (Pua, 1993). In
B. campestris (Chi et al., 1994) there was an increase Vegetative growth
in the endogenous levels of free put and spm in the
presence of AVG, together with the promotive effect of Exogenous PAs were found to affect metabolism in
exogenous PAs on shoot regeneration suggesting that pine callus but did not prevent browning and deteri-
cell differentiation leading to shoot formation from oration of the callus cultures (Laukkanen and Sarjala,
cotyledons may be associated with polyamine meta- 1997).
bolism. This result indicates that the promotive effect In rice coleoptiles ADC was found to mediate the
of PAs in regeneration may not be due to an inhibition ethylene induced Put accumulation. There was an in-
of ethylene biosynthesis. crease in SAMdc activity but no significant Spd/Spm
In rice callus there is a loss of regeneration po- accumulation. DFMA inhibited the ethylene induced
tential with increasing age. Twelve-month-old cultures Put accumulation and coleoptile elongation (Lee and
showed a 5-fold increase in Put and a 2.5-fold increase Chu, 1992). IAA stimulated the coleoptile elonga-
in Spd levels (Bajaj and Rajam, 1995). The long-term tion and Put accumulation in rice. DFMA, but not
cultures exhibited massive accumulation of Put and DFMO was found to inhibit the IAA stimulated co-
Spd along with an increase in ADC activity and the leoptile elongation and Put accumulation. Addition of
near lack of morphogenetic capacity in such cultures Put could not reverse the effect of DFMA (Lee and
could be completely revived by blocking the PA ac- Lin, 1996).
 13

In saffron corms, free Put was not detected at the been given to the involvement of PAs in fruit develop-
onset of sprouting, whereas free Spd and Spm levels ment and senescence. Two sorts of claims have been
increased rapidly on sprouting and decreased during made for PAs. One is simply that exogenous applic-
further stages of corm development. The levels of con- ation will retard senescence. The second is that the
jugated PAs were several times higher than the free lowering of the polyamine concentration is an import-
forms indicating their possible role in the develop- ant early step in triggering senescence. Also bearing
mental processes. A comparison of polyamine levels on these two hypotheses is the question of whether
of vegetative and floral corms showed higher titers of exogenous application of PAs causes novel physiolo-
free PAs in vegetative and those of conjugated PAs in gical effects or performs the normal function of the
floral corms (Jirage et al., 1994). Similar results have endogenous pools (Evans and Malmberg, 1989).
been obtained in tobacco (Smith, 1985). Galston et al. (1978) et al. (Altman et al., 1977;
Kaur-Sawhney et al., 1978, 1982; Kaur-Sawhney and
Interaction with secondary metabolites Galston, 1979) have reported an anti-senescence effect
of PAs on excised oat leaves incubated in the dark. A
Hydroxy-cinnamic acids conjugate with PAs and these short exposure of the excised leaves to a buffer con-
complexes are widespread in the plant kingdom, par-
taining a polyamine significantly retarded senescence
ticularly in the Solanaceae (Smith et al., 1983). Thin
as measured by retention of chlorophyll and inhibition
epidermal layers of tobacco cultured on a rhizogenic of RNAase and protease activities. Similar observation
medium exhibited strong alterations in their wall char-
has been made on detached leaves and cell cultures of
acteristics associated with cell hypertrophy and sep-
variety of monocot and dicot species (Kaur-Sawhney
aration (Berta et al., 1997). The tobacco cell walls and Galston, 1979; Srivastava et al., 1983). The ef-
are particularly rich in ester-linked phenolic acids, es-
fect of the exogenous polyamine applications is thus
pecially hydroxycinnamic acids, such as ferulic acid, similar to those of exogenous cytokinin, although cy-
p-coumaric acid and dehydrodiferulic acids, whose tokinins are typically applied at 0.1 mM and PAs are
covalent cross-links to arabinoxylans and/or pectic
applied at roughly 10 mM.
polysaccharides could be involved in cell extension Cheng et al. (1984) have studied senescence of de-
(Liyama et al., 1994). The specific Spd-binding activ-
tached rice leaves and have suggested the involvement
ity with total proteins solubilized from plasma mem-
of diaminopropane in the results obtained with exo-
brane from Cucurbita pepo L. was studied. A specific genously applied Spd and Spm. All three appeared to
PA interaction between Spd and plasma membrane
retard senescence in the dark and all promoted chloro-
occurs with the protein component of the membrane phyll degradation in the light. However, the presence
(Tassoni et al., 1996, 1998). Diamines and Put are or- of α-hydroxyethylhydrazine, an inhibitor of conver-
ganic polycations that adsorb differentially on plant on
sion of PAs to diaminopropane by polyamine oxidase,
plant cell wall (Messiaen et al., 1997). Messiaen and reversed the effect of Spm and Spd in light, indicating
Cutsem (1999) have shown that PA binding to pec-
a possible requirement for conversion to diaminopro-
tin oligomers modulates/pectin signal these oligomers
pane. Ca2+ was shown to inhibit the observed effects
generate in plant cells. PAs seem to be essential for competitively, as discussed in previous section (See
conferring normal size and rigidity on the primary cell
Cell Division) suggesting that an initial attachment of
wall, by favoring cross-links between wall compon- the PAs to a membrane site may be required (Cheng et
ents and probably increasing their synthesis. Biondi al., 1984).
et al. (2000) have reported that the treatment of root
The mechanism of inhibiting senescence by exo-
cultures of Hyoscyamus muticus with jasmonates in- genous PAs may be related to their possible inhibition
creased the putrescine and methylputrescine levels and
of ethylene synthesis (Apelbaum et al., 1981, 1985)
moderately increased the tropane alkaloid levels. A
and to stabilization of membranes (Grimes et al.,
possible interrelationship between the biosynthesis of 1986). An observation that supports an involvement of
PAs and that of tropane alkaloids has been suggested.
membrane stabilization is that, the addition of Ca2+
ions will counteract the ability of PAs to stabilize
Senescence
chlorophyll levels in senescing leaves (Kaur-Sawhney
Because of the potential metabolic interaction/compet- and Galston, 1979). Fuhrer et al. (1982) found in
ition between polyamine and ethylene biosynthesis peeled oat leaves that exogenous PAs repressed ethyl-
through the use of SAM, a great deal of attention has ene synthesis, particularly the conversion of ACC to
14

ethylene. They suggested that exogenous PAs initially cific effects frequently reported for exogenous PAs.
attach to membranes and then inhibit ethylene produc- In contrast, Dibble et al. (1988) measured polyamine
tion and retard senescence. In agreement with this, levels in a landrace of tomato (alcobaca) that ripens
Drolet et al. (1986) have found significant free rad- more slowly and had better storage characteristics.
ical scavenging by PAs correlated with the number of This could be compared to a mutation isolated from
amino groups. They suggested this might be part of alcobaca that had lost the storage and ripening charac-
the membrane stabilization and senescence retardation teristics (in a sense, a revertant); the mutant thus had
observed in various systems. They also noted that the the developmental timing seen in most other tomato
conversion of ACC to ethylene is superoxide depend- cultivars. The tomato fruit showed a clear increase in
ent, and that this reaction was inhibited by PAs. There Put content, especially at later stages of development,
is thus a reasonable working model for the inhibition compared to normal tomato. These genetic data thus
of senescence by exogenous PAs, involving binding provide significant additional evidence that polyam-
to membranes, prevention of lipid peroxidation, and ine levels are inversely correlated with senescence and
quenching of the free radicals needed for the ACC to that large endogenous pools of PAs may have effects
ethylene conversion. similar to those of exogenous polyamine applications.
Roberts et al. (1986) presented evidence with mi- Several examples are known where PAs did not
crosomal membranes from Phaseolus that exogen- seem to retard senescence. Downs and Lovell (1986)
ously applied PAs associate with membrane lipids found that addition of Put and Spd, (at 10 mM), to
and substantially reduce membrane fluidity. Agazio culture solutions of cut carnations, could actually res-
et al. (1988) found that PAs could inhibit washing- ult in greater ethylene production and reduced bloom
stimulated K+ influx and H+ efflux without interfering longevity. Roberts et al. (1984) had reported that endo-
with K+ uptake and H+ efflux stimulated by fusicoc- genous Put levels rose during senescence of carnations
cin. They argued that this differential effect is evidence without apparent inhibition of ethylene, but that inhib-
that polyamine effects on membranes are specific at ition of ethylene with aminoxyacetic acid resulted in
physiological concentration. increase in Spd in petal tissues. This increase in Spd
A distinctly different mechanistic hypothesis is would be predicted if there were channeling of SAM
that the senescence signal itself acts by decreasing to the polyamine pathway because of blockage in the
PAs as one of the critical control steps. Evidence ethylene pathway (Even-Chen et al., 1982). Srivastava
countering the role of polyamines in control of senes- et al. (1983) compared endogenous polyamine levels
cence was provided by Smith and Davies (1985), who in light and the dark grown barley seedling and sen-
worked with a photoperiod inducible senescence in the escing leaves. The levels were comparable under all
apical bud of peas, a property of two dominant alleles, conditions with the possible exception of a decline
Sn and Hr. They found during bud senescence that the in Spd with senescence. Application of kinetin sup-
amounts of PAs per organ declined with the decreased pressed the decline of Spd. Furthermore, exogenous
size of the bud, but there was no decrease of PAs prior PAs did not retard senescence in the dark; however,
to the appearance of early symptoms of senescence. Spd and Spm treatment in the presence of light en-
They concluded that PAs were not part of the chain hanced retardation of senescence. In unpollinated pea
of events leading from the photoperiodic signal to the ovaries, onset of senescene is preceded by a decrease
initiation of senescence. in Put and Spd content and by an increase in Spm
An important question is whether or not exogenous titers. Furthermore Spm levels decreases when fruit
PAs affect senescence the same way as the internal growth is induced by application of growth regulators
pools do. Birecka et al. (1989) compared chloro- such as auxins, GA3 or by pollination (Carbonell and
phyll and protein degradation of Avena, Nicotiana, Navarrno, 1989).
and Heliotropium detached leaves in the dark. They Although there are a few counter examples, exo-
found that older Heliotropium leaves with very low genous PAs retard senescence in many species and
polyamine levels exhibited only a weak senescence experimental system. Senescence of Petunia hybrida
syndrome, whereas the leaves of the other two species flowers is accompanied by a climacteric pattern in
had high levels of PAs and exhibited pronounced sen- ethylene production and a rapid decline in the levels
escence with no significant polyamine decline. These of Put and Spd during the preclimacteric phase (Botha
observations led them to suggest that endogenous PAs and Whitehead, 1992). The decrease in Spd is caused
may have effects different from the possibly nonspe- by the decline in the availability of Put, which is ini-
 15

tially synthesized, from L-arginine via agmatine and Modulation in polyamine biosynthesis by plant
N-carbamoylputrescine. Inhibition of Put and PA syn- growth regulators
thesis resulted in a concomitant increase in ethylene
production. In unpollinated flowers the onset of the There is considerable evidence that polyamine bio-
climacteric rise in ethylene production was acceler- synthesis in animals is responsive to hormones and
ated after treatment with PAs. However, in pollinated other activator molecules (Perella et al., 1983; See-
flowers this process was delayed as a result of treat- ley et al., 1982, 1983). In fact, a report by Koenig et
ment with low concentration of PAs. Based on the al. (1983), documenting rapid (<30 s) ODC activa-
effects of exogenous PA on ethylene production in tion and increased polyamine biosynthesis following
both pollinated and unpollinated flowers Botha and testosterone binding to mouse kidney cells, suggests
Whitehead, (1992) concluded that ethylene synthesis that the polyamine biosynthetic machinery is exquis-
in these flowers is not regulated by a feedback control itely sensitive to such hormonal regulation.
mechanism (Botha and Whitehead, 1992). In plants, growth regulatory hormones have also
been shown to elicit tissue specific changes in polyam-
ine metabolism. It has been proposed that PAs, which
Regulation of polyamine biosynthesis
may or may not be mobile in plants (Young and Gal-
ston, 1983a), may serve as in mediating intracellular
Many aspects of plant growth and development are
hormonal effects (Galston, 1983). Some evidence sup-
correlated with changes in polyamine metabolism.
porting this hypothesis has been obtained through the
Moreover, most important growth and developmental
use of specific inhibitors of polyamine biosynthesis
parameters are also known to be regulated by plant
(Bagni et al., 1978).
hormones, perhaps it is not surprising that many hor-
In dormant Helianthus tuberosus tuber tissue,
monal responses in turn are accompanied by dynamic
where endogenous polyamine levels appear to limit
changes in PA metabolism and is associated with cer-
growth, the application of an auxin, 2,4-D, activated
tain photomorphogenic responses. Consequently, PA
endogenous polyamine biosynthesis, macromolecular
metabolism appears to be involved in the physiology
synthesis, and growth (Bagni et al., 1978). Exogen-
of growth regulation.
ous PAs can substitute for the auxin in this response
Phytochrome control of PA biosynthesis (Bagni et al., 1978), further supporting the idea of
polyamine-mediated hormonal responses. Similarly,
In plants, the photoreceptor chromoprotein phyto- the promotion of parthenocarpic fruit development in
chrome, regulates many aspects of plant growth and tomato by auxin is characterized by a rapid increase
development and studies have supported the view that in ODC activity (Mizrahi and Heimher, 1982). Since
one probable locus of activity is the regulation of ADC normal post-fertilization growth in this organ, which
(Dai and Galston, 1981). Irradiation of etiolated pea is induced by endogenous auxin, can be specifically
seedlings with actinic (red) light was found to decrease inhibited by DFMO and is reversible by exogenous
ADC activity and elongation in internodes, while sim- Put, polyamine biosynthesis appears to be prerequis-
ultaneously increasing ADC activity and growth in the ite for hormone-mediated growth in these tissues as
terminal bud (Dai and Galston, 1981). In pea buds well (Mizrahi and Heimher, 1982). Likewise, auxin-
also, photoreversible control of ADC activity was ob- induced adventitious root formation in mung bean
served, which indicates that the promotion of ADC seedlings involves increased polyamine biosynthesis
activity in buds is mediated by phytochrome, as is (Friedman et al., 1982). Inhibition of this response by
its inhibition in epicotyls (Dai and Galston, 1981). In MGBG, and its reversal by the application of arginine
plants, increasing evidence suggests that ADC activ- or ornithine, again provides evidence that polyamine
ity as well as PA levels are elevated in growing tissue biosynthesis is required in this response (Friedman et
(Suresh et al., 1978; Montague et al., 1979). It seems al., 1982).
clear, however, that changes in polyamine metabolism Other plant growth and developmental responses
are not simply a consequence of altered growth rates, to hormones appear to involve changes in polyam-
since researchers have been able to demonstrate a sep- ine metabolism as well. It has been shown that
aration of ADC activity from either light (Goren et al., gibberellin promotion of elongation of dwarf pea in-
1982b) — or hormonally — induced (Palavan et al., ternodes is accompanied by simultaneous increases
1984) changes in growth rates in etiolated pea stems. in polyamine titers and ADC activity (Dai et al.,
16

1982). Cho (1983) also reported that Put synthesis ADC activity. Icekson et al. (1985) similarly showed
increases during GA3 -induced elongation of lettuce that ethylene reduced SAMdc activity in pea apices.
hypocotyls. The gibberellin-induced synthesis of α- The same authors subsequently reported in the same
amylase in barley aleurone tissue can be inhibited by experimental system that lysine decarboxylase activ-
DFMA (Bernal-Lugo, 1983), although the nature of ity increased and cadaverine levels rose in response
any polyamine involvement in this response is not to ethylene administration. They speculated that this
yet clear. Lin (1984) reported that polyamine titers might be a compensation for the decrease in ADC.
in aleurone layer cells are not altered in response In peeled oat leaves, Spd and diaminopropane were
to either GA3 promotion or abscissic acid inhibi- shown to reduce ethylene levels both by inhibition of
tion of α-amylase. However, prior depletion of Spd ACC synthase and by conversion of ACC to ethylene
titers, by MGBG inhibition of SAMdc, decreases the (Fuhrer et al., 1982). A reduction in chlorophyll loss
subsequent GA3 induction of α-amylase activity, sug- also occurred that was not caused by lowered ethyl-
gesting that PAs may play some role in the hormonal ene levels. Since Ca2+ addition could competitively
induction of this enzyme. Kyriakidis (1983) has shown reduce these polyamine effects it was suggested that
that both GA3 and IAA promote a 4-fold increase a membrane attachment by the PAs was responsible
in ODC activity in germinating barley seedlings. Cy- for the effects. However using apple disks and cor-
tokinins have also been reported to activate polyamine relating microsomal membrane microviscosity with
biosynthesis in a few plant systems. Kinetin and ben- ethylene production Ben-Arie et al. (1982) showed the
zyladenine increased Put synthesis in cotyledons of inhibitory effects of Ca2+ and Spm to be temperature
light-grown lettuce (Cho, 1983). Suresh et al. (1978) dependent and to have very different curves. They con-
reported that ADC activity and Put contents increased cluded that the modes or sites of action of the Ca2+
in response to benzyladenine treatment. Cytokinins and Spm might differ.
were also able to reverse the inhibition of this response Ke and Romani (1988) showed that induced ethyl-
by another plant hormone, abscissic acid, possibly in- ene production in suspension cultures of pear fruit
dicating that polyamine biosynthesis in these seedlings cells indicated that Spd at 1 mM had to be applied
is under the control of multiple hormones. ADC activ- prior to ethylene induction in order to have an effect on
ity in radiated, etiolated pea buds was also increased ethylene appearance; the application also resulted in
by low concentrations of benzyladenine, which did not reduced polysome numbers. Put and Spm had similar
affect growth (Palavan et al., 1984). effects. These results were interpreted to mean that the
Inhibitors of both polyamine and ethylene syn- suppression of ethylene production might have been
thesis make it possible to probe the interaction of PAs caused by suppression of macromolecular synthesis
with ethylene in a variety of experimental procedures. with subsequent lower levels of ACC synthase and
The most common hypothesis tested is that PAs and ACC.
ethylene may regulate each other’s synthesis, either The use of the polyamine synthesis inhibitors on
directly or by metabolic competition for SAM. In ad- cut carnations increased their rate of ethylene pro-
dition, Miyazaki and Yang (1987) have pointed out duction and senescence, while the application of an
that PAs and ethylene biosynthesis must both allow for ACC synthase inhibitor decreased ethylene production
recycling of methylthioadenosine and that this process and increased polyamine levels (Roberts et al., 1984).
may be as significant as the more frequently studied Even-Chen et al. (1982) used [3,4-14C]methionine
fate of the propylamine group from SAM. to trace carbon flow into either ethylene or PAs
PAs inhibit ethylene formation in a number of plant in aged orange peel discs. A variety of treatments
tissues, including apple fruits, bean and tobacco leaf were applied to inhibit ethylene synthesis, including
explants (Apelbaum et al., 1984), Tradescantia petals, phosphate/Co2+, aminoethoxyvinylglycine, and exo-
and mung bean hypocotyls (Suttle, 1981). Apelbaum genous Put. These treatments resulted in a 3–4-fold
et al. (1985) have measured ADC activity in the ap- increase in the specific label of Spd, suggesting that
ical meristem of peas. In response to ethylene, the sharing a precursor pool of SAM connects the two
levels of ADC activity decreased by 90% within 18 pathways. Unlabeled Spd inhibited transfer of label
h; ethylene also increased the Km and decreased the to ACC but did not stimulate label incorporation into
Vmax of ADC. Reducing endogenous ethylene with hy- spermidine; the spermidine inhibition of label trans-
pobaric pressure or treatments with silver thiosulfate fer to ACC was partially overcome by the addition of
and 2,5-norbornadiene led to 30–50% increases in Ca2+ , a result consistent with earlier senescence stud-
 17

ies (Fuhrer et al., 1982; Kaur-Sawhney and Galston, sativus), wherein they have reported that exogenous
1979). putrescine adminstration intervened with total ethyl-
Two alternative forms of interaction between PAs ene production and was a keyfactor in regulating
and ethylene biosynthesis have been proposed. Drolet morphogenesis.
et al. (1986) demonstrated that Spm, Spd, Put and Cad In our laboratory working with normal and trans-
could effectively act as scavengers of free radicals, al- genic shoot cultures of chicory, we found that there
though at the relatively high concentrations of 10 and exists a competitive utilization of the common pre-
50 mM, and could inhibit the superoxide-dependent cursor SAM between these two pathways. Exogenous
conversion of ACC to ethylene. Winer and Apelbaum feeding of Put resulted in reduced production of ethyl-
(1986) suggested that PAs might inhibit ACC synthase ene (Bais, 2000). Furthermore, these studies have
by forming a Schiff base with its cofactor, pyridoxal shown that administration of AgNO3 to the shoot
phosphate. No conclusive evidence for this proposal cultures of chicory resulted in morphogenesis with
has yet appeared. a lesser production of ethylene (Bais et al., 2000b).
Counter examples exist in which ethylene and PAs The intervention of ethylene biosynthesis by addition
are not mutually antagonistic. A study of chilling ef- of exogenous Put was confirmed by polyamine bio-
fects on cucumber seedlings has shown that prevention synthetic inhibitor studies wherein increased ethylene
of ACC induction by aminooxyacetic acid does not production was observed probably due to opening of
raise polyamine levels and that increases in Spd and the pathway and over-utilization of SAM for ethylene
ACC are stimulated simultaneously by the chilling biosynthesis (Bais et al., 2000b).
treatment. These observations imply that regulation Of the major plant hormones, ethylene has been the
may not occur at the level of SAM competition (Wang, most intensively investigated with respect to polyam-
1987). Similar situations in which the formation of ine metabolism. This can largely be attributed to the
ACC and the formation of polyamines increase simul- fact that PAs exhibit marked anti-senescence proper-
taneously occur in apple and cherry buds (Wang et al., ties (see ethylene intervention), directly antagonizing
1985; Wang and Steffens, 1985). Kushad et al. (1988) many ethylene-mediated responses with SAM being a
measured polymines and ethylene during avocado fruit common intermediate to both ethylene and PAs.
development and found that PAs peaked earlier than The question of major interest in these studies is
ethylene. They suggested there was no competition how PAs and ethylene, which inhibit or promote sen-
between the two pathways, although their results did escence, respectively, interact at the cellular level.
not exclude the possibility of metabolic competition Fuhrer et al. (1982) have found that exogenous PAs
during the peak phase of polyamine synthesis. inhibit ethylene biosynthesis by blocking the conver-
Cohen and Kende (1986) have examined the sion of ACC to ethylene, an event that is catalysed
polyamine synthetic enzymes in deep-water rice. Sub- by a constitutive membrane-associated enzyme sys-
mergence or treatments with gibberellic acid or ethyl- tem (Mattoo et al., 1977). Partial calcium antagonism
ene stimulate increased cell division and elongation of the PA inhibition of ethylene synthesis in senes-
in the intercalary meristems of these plants. They cing leaf tissues (Apelbaum et al., 1981; Fuhrer et al.,
found a 2-fold increase in ADC activity and an 8- 1982) suggests that PAs mediate this response through
fold increase in SAMdc activity peaking at 8 h after ionic interaction with membranes and, presumably, by
submergence; putrescine levels rose 4-fold and sper- modulating the activity of the ethylene-synthesizing
midine levels rose 2-fold. Treatment of isolated stem complex (Ben-Arie et al., 1982). Even-Chen et al.,
sections in air with either ethylene or GA3 also resul- (1982) and Fuhrer et al. (1982), indicated that exogen-
ted in 2–4-fold increases in ADC and SAMdc activ- ous PAs block the formation of ethylene by inhibiting
ities, although no change was detected in polyamine the production of its ACC synthase. Of great interest
levels. Neither treatment affected ODC. These results was the discovery by Even-Chen et al. (1982) that
thus contrast with those showing ethylene and PAs this block in ACC synthesis was accompanied by
to be mutually inhibitory or antagonistic. In this sys- increased incorporation of methonine derived SAM
tem, ethylene stimulates both growth of the intercalary into PAs. Thus, the anti-senescence properties of
meristem and ADC and SAMdc activities. Pua et al. exogenous PAs appear to result from a simultaneous
(1996) have reported the synergistic activity of ethyl- inhibition of ethylene biosynthesis and prohibition of
ene inhibitors and putrescine on shoot regeneration ethylene biosynthesis and promotion of their own syn-
from hypocotyl explants of chinese radish (Raphanus
18

thesis, through interrelated pathways, which may be Since the original report by Richards and Coleman
subject to feedback regulation. (1952), a number of workers have documented large
Roberts et al. (1984) have provided convincing increase in Put titers in a variety of plants experien-
evidence to support this view, using specific inhibit- cing potassium deficiency (Smith, 1970; Klein et al.,
ors of both polyamine and ethylene biosynthesis. In 1979; Young & Galston, 1984). Murty et al. (1971)
senescing carnation flowers, inhibition of polyamine suggested that the increase in Put levels compensated
biosynthesis by DFMA or MGBG leads to increased for approximately 30% of the K+ deficit in black cur-
rates of ethylene production and onset of senescence. rant leaves, and thus might function in maintaining an
Conversely, inhibition of ACC synthesis from SAM ionic balance in these tissues.
by α-aminooxyacetic acid, an inhibitor of ACC syn- Acid feeding (Smith and Sinclair, 1967; Young and
thase, decreases ethylene production and senescence Galston, 1983b), exposure to SO2 (Priebe et al., 1978),
is delayed. Of particular interest, however, was their or NH4+ nutrition (Priebe et al., 1978; Klein et al.,
finding that the inhibition of ethylene synthesis by α- 1979) and all treatments which generate excess H+
aminoisobutyric acid, which blocks the conversion of ions in the tissue, also produce elevated Put levels in
ACC to ethylene, had no significant effect on polyam- plants. There was a marked difference in the PA levels
ine biosynthesis. By extrapolation, one would assume in healthy and diseased trees of Picea in acid-rain af-
that ACC synthase and aminopropyltransferase activ- fected areas depending on the physiological state of
ities, likewise, are not involved in feedback regulation the trees (Santerra et al., 1990). The key rate-limiting
of polyamine biosynthesis in this plant. It is known enzyme in the conversion of arginine to put in plants is
that the activity of purified spermine synthase from ADC, the activity of which increases in different kinds
bovine brain is greatly inhibited by methylthioaden- of ionic stresses (Smith, 1963, 1984; Smith and Sin-
osine (MTA) (Pajula and Raina, 1979). Thus, this clair, 1967), particularly with acidic stress (Young and
metabolite would seem to be an attractive candidate Galston, 1983b). The treatment of bean plants with
in a feedback inhibition scheme involving ethylene simulated acid rain induced lipid peroxidation and in-
and polyamine biosyntheses. It is known, however, creased level of H2 O2 in the leaves. Pretreatment with
that MTA levels in plant tissues remain relatively low, spd and spm prevented these changes (Velikova et al.,
due to MTA nucleosidase activity (Guranowski et al., 2000).
1981). To substantiate this, Giovanelli et al. (1981) A similar response seen in osmotically stressed
have proposed that a primary function for the efficient tissues (Flores and Galston, 1984a, 1984b), may rep-
metabolism of MTA in Lemma is the facilitation of resent a mechanism for regulating water losses by
polyamine biosynthesis. Rugini (1992) observed that increasing intracellular osmolarity, appears to be fun-
there exists a synergistic activity between PAs and damentally different from the responses of bacteria
auxins, in A. rhizogenes mediated in vitro rooting in (Munro et al., 1972) and mammalian cells (Munro et
olive species. al., 1975). In these organisms, increased osmolarity
In summary, it seems clear that hormone action and produces a rapid excretion of Put in response to K+
synthesis (in the case of ethylene) involves changes uptake, resulting in a depletion of intracellular Put
in PA metabolism of these hormone responses. Un- levels.
doubtedly, active investigation in this exciting area of The gradual accumulation of Put in K+ deficient
plant physiology will continue. plants (Smith, 1979; Young and Galston, 1984), or
in plants maintained with NH4+ as a nitrogen source
(Young and Galston, 1983b) was correlated with in-
Polyamine metabolism and plant stress creased ADC activity over a period of days or weeks.
In K+ -deficient oat leaves, ADC levels were approx-
Many types of stress conditions produce character- imately 8-fold higher than controls, and this tissue
istic metabolic changes during which Put accumulates served as a convenient source for the purification of
to high concentrations in plant tissues, while other ADC (Young and Galston, 1983b). Increased Put
polyamine levels remain essentially unchanged. Al- titers in H+ stressed (Young and Galston, 1983b)
though the physiological significance of this response and osmotically stressed (Flores and Galston, 1984a,
is not understood, it may be of fundamental import- 1984b) oat leaves were also correlated with increased
ance in view of the fact that plants growing in natural ADC activities. In both instances, response developed
environments must continuously adapt to grow. within 1–2 h and was dependent upon continued pro-
 19

tein synthesis, as indicated by cycloheximide inhibitor elicit the production of hydroxycinnamic acid amide
studies. The increase in Put titers, which may be as conjugates of 2-hydroxyputrescine, an unusual meta-
much as 60-fold higher in osmotically stressed leaves bolite that may function as a phytoalexin (Samborski
than in control leaves after 6-h treatment (Flores and and Rohringer, 1970). In barley seedlings, the pro-
Galston, 1984a), can be blocked by DFMA, but not duction of hordatines (dicoumaryoylagmatines) com-
by DFMO. These results suggest that Put synthesis is pounds with mild antifungal activity, increases upon
primarily the result of increased ADC activity, rather fungal infection (Smith and Best, 1978). Stroinski and
than ODC activity, in the stressed plant. Szezotka (1989) have shown that cadmium stress and
Little is known regarding the physiological signi- infection upon Phytopthora infestans resulted in 2-
ficance of Put accumulation, in response to stress in fold increase in endogenous polyamine titers in potato
plants. The response may be of a protective nature, cell suspension cultures.
conferring a selective advantage to the stressed cells. Arabidopsis thaliana responds to potassium de-
The anti-senescence properties of exogenous PAs have ficiency stress by increasing ADC activity 10-fold
already been discussed, but it is not known whether and by elevating Put levels 20-fold (Watson and
these PAs enter senescing cells and exert their effects Malmberg, 1996). Young and Galston (1984) repor-
at the intracellular level. Diamines, such as Put, are ted that potassium deficient oat plants exhibited a
generally less effective than PAs (Spd, Spm) in pre- 6-fold increase in Put levels after 18 days of growth.
venting increases in RNAase and protease activity, Khan and Harborne (1991) reported that potassium
chlorophyll degradation, and other processes culmin- deficiency could increase Put levels and tropane al-
ating in senescence (Kaur-Sahwney et al., 1978, 1982; kaloid synthesis in Atropa acuminata. Put can also
Kaur-Sahwney and Galston, 1979). Put is also less be a precursor for pyrrolidine alkaloids (Tiburcio et
effective than PAs in stabilizing membranes against al., 1990). Exogenous Put application was found to
stress-induced changes in fluidity (Ben-Arie et al., increase oxidant resistance in Conyza bonariensis (Ye
1982) and solute leakage (Altman, 1982). Thus, the et al., 1997).
failure of Put to be metabolised to Spd and Spm The ODC activity and PA contents were found
would not appear to be advantageous from an anti- to increase significantly in wheat leaves exposed to
senescence point of view. To the contrary, Put accu- ozone and hydrogen fluoride. The increase in ODC
mulation could be a cause of stress-induced injury. activity may be a mechanism to increase the PA levels
High concentrations (∼100 mM>) of exogenously ap- in leaves exposed to such gases so as to minimize the
plied Put are reported to be toxic to number of plant damaging effects (An and Wang, 1997).
tissues (Shevyakova, 1966), and large accumulations In wheat, addition of K+ inhibited the ABA-
of endogenous Put may be related to leaf necrosis in mediated increase in growth and Put levels. Put in-
virus-infected (Torget et al., 1979) and K+ -deficient crease induced by ABA was inhibited by both DFMA
(Basso and Smith, 1974) leaf tissues. Clearly, the in- and DFMO in shoots while only DFMA was effective
tracellular accumulation of high concentrations of free in the root (Aurisano et al., 1994).
amines, such as Put, would have drastic consequences There was a decrease in ODC activity in response
for the regulation of nitrogen metabolism, protein syn- to citrus viroid infection or ethephon treatment in
thesis, and the maintenance of cellular pH and ion tomato plants. ADC activity was not altered. Cit-
homeostasis. rus viroid infection had no effect on PA conjugates;
Such considerations may be relevant to the prob- and ethephon produced a decrease in the PA con-
lem of the regeneration of cereal plants from pro- jugates. Interference with ethylene action by silver
toplasts. In light of various types of environmental ions prevented the decrease in ODC activity and in
stress-induced alterations in polyamine metabolism free and conjugated Put. It is suggested that changes
discussed above, perhaps it is appropriate here to men- in Put levels after citrus viroid infection and ethep-
tion that numerous plant pathogens elicit changes in hon treatment are regulated via ODC activity and that
polyamine metabolism in the host tissues, as well. In conjugation is not involved (Belles et al., 1993).
brown rust-infected barely leaves, chlorophyll is re- The levels of PAs and ADC decreased in Heli-
tained in so-called ‘green island’ at infection sites in anthus annus seedlings in response to salt stress. ODC
the senescing leaves, and Spd levels in these tissues in- activity was not detected. The variation in Put content
crease six-to seven fold (Greenlands and Lewis, 1984). was supposed to be entirely due to the decrease in
In resistant wheat cultivars, a variety of pathogens ADC activity. DFMO and DFMA did not inhibit but
20

delayed the onset of germination of sunflower seeds, In our laboratory, working with hairy root cultures
and DFMO increased the content of Spd and Spm. of C. intybus fungal elicitation for growth and en-
This suggests that PAs could be involved in the ger- hancement of coumarins, we observed, an increase in
mination process of H. annus seeds and in response to total endogenous polyamine titers (Bais, 2000).
salt stress (Benavides et al., 1997). All of these studies suggest that changes in
The synthesis of ADC in osmotically stressed oat polyamine metabolism may play a role in plant adapt-
leaves is regulated by Spm. Treatment with Spm in ation to agents inducing biological stress.
combination with osmotic stress was reported to res-
ult in increased steady-state levels of ADC mRNA,
with a decrease in ADC activity (Borrell et al., 1996). Biocontrol of plant disease and polyamines
This absence of correlation is explained by the fact
that Spm inhibits processing of the ADC proenzyme, Inhibition of fungal polyamine biosynthesis
which results in increased levels of this inactive ADC
form and a consequent decrease in the ADC-processed Since PAs play a crucial role in cell proliferation and
form. Spm treatment leads to delayed loss of chloro- differentiation, the selective inhibition of polyamine
phyll in dark-incubated and osmotically stressed oat biosynthesis has provided a novel and promising ap-
leaves. Thus the post-translational regulation of ADC proach for new therapies. In other words, all the
synthesis by Spm may be important in explaining its enzymes for polyamine biosynthesis have been targets
anti-senescence properties (Borrell et al., 1996). for disease prevention (McCann et al., 1987; Palfrey-
In salt-tolerant rice cultivar Pokkali, ADC enzyme man et al., 1989). The most exciting and far-reaching
activity increased and its transcripts also accumulate development in this area is the recent possibilities
during prolonged salinity stress, this mechanism is of control of fungal plant diseases through specific
absent in the salt-sensitive rice cultivar where a pro- inhibition of polyamine biosynthesis are interesting
longed period of salinity stress downregulates both applications (Rajam et al., 1985; Walters and Wylie,
ADC activity and its transcript level (Chattopadhyay 1986; Rajam, 1987; Walters, 1987, 1989; Galston and
et al., 1997). Weinstein, 1988). Fungal pathogens that cause dis-
Long-term salinity stress in Brassica campestris eases in higher plants also need PAs for normal growth
caused only small changes in PA levels, ADC, ODC and differentiation (Rajam and Galston, 1985), but
and PAO activities. DAO activity, unlike other PA unlike plants they have but a single pathway (ODC)
metabolic enzymes, increased significantly during for the biogenesis of PAs (Tyagi et al., 1981; Tabor
long stress (Das et al., 1995). Put was found to be and Tabor, 1985). It should be possible, therefore, to
essential for the recovery of growth in chilled roots of eradicate a fungal pathogen on a crop plant without
rice (Lee, 1997). Bagga et al. (1997) reported higher affecting the growth of host plant by specific inhibition
activity of aminopropyl transferase (APT) in osmotic of fungal ODC pathway (Rajam and Galston, 1985;
stress tolerant strains of alfalfa. This suggests that pu- Rajam et al., 1985).
trescine aminopropyl transferase (PAPT) is present in The exogenous application of Put caused a sharp
alfalfa, which is subjected to osmotic stress wherein decline in microcycle conidiation in Aspergillus flavus
Put is utilized as sole initial substrate (Bagga et al., in a dose-dependent manner but induced vegetative
1997). Mansour and Al-Mutawa (1999) have reported growth. PA inhibitors like DFMO, MGBG and CHA
that the total endogenous PAs plays an important role resulted in complete inhibition of microcycle conidi-
in stabilization of plasma membrane under salt stress. ation at 5 mM levels. The inhibitory effect of PA
In rice seedling methyl jasmonate induced chilling inhibitors was partially reversed by exogenous Put or
tolerance, which was reduced by DFMA. The effects Spd, with Spd being more effective than Put. Put has
of DFMA were partly prevented by Put. This indic- been implicated to be essential for vegetative growth,
ates that Put accumulation is required for the induction while Spd is involved in microcycle conidiation and
of chilling tolerance in rice seedlings by methyl jas- a low Put/Spd ratio seems to be important for spore
monate (Lee et al., 1996). Treatment of bell pepper differentiation to microcycle conidiation (Khurana et
fruits with hot water in conjunction with film pack- al., 1996).
aging may delay chilling injury and decay through a
mechanism that involves elevation of polyamine levels
(Gonzalez-Aguilar et al., 2000).
 21

Inhibition of plant pathogenic fungi in vitro Control of fungal plant diseases

Rajam and Galston (1985), for the first time demon- DFMO (0.5 mM or higher) is effective protectant
strated that DFMO at 0.1, 0.5 and 1.0 mM strongly against the bean rust fungus, Uromyces phaseoli (Ra-
inhibited the growth of several phytopathogenic fungi jam et al., 1985). DFMO conferred protection against
in vitro, and such inhibitions can be totally reversed infection even in unsprayed regions of the bean plant,
by exogenously supplied PAs. This also indicated the suggesting translocation of the compound or some
absolute requirement of PAs for normal growth and other inhibitory substance as naturally occurring an-
development of fungal hyphae. DFMO and DFMA timicrobial agent produced as a result of DFMO treat-
can cause changes in mycelial morphology and cell ment in the host plant (Rajam et al., 1985; Slocum,
size. Cell length was much reduced and cell diamet- 1991). PAs alone did not affect the rust infection of
ers were increased in inhibitor-treated cultures, while beans, however, when Spd was supplied 1 h after
considerable increase in cell length and diameter was DFMO, the inhibition conferred by DFMO was sub-
observed in many PA-treated cultures (Rajam and Gal- stantially reduced (Rajam et al., 1986). This suggests
ston, 1985). West and Walters (1989) showed that the that the effect of DFMO was related to inhibition of PA
cell size of G. graminis barely changed, but some biosynthesis. The control of several other fungal plant
treatments produced a decrease in cell length. Pyr- diseases such as the common rust of corn, southern
enophora teres also showed reductions in lengths on corn leaf blight, oat stem rust, apple powdery mildew
exposure to several inhibitors. has been achieved in field conditions using DFMO
Foster and Walters (1990) reported that neither and/or DFMA (Galston and Weinstein, 1988).
the PA inhibitors nor exogenous PAs had any sig- The control of some infections by DFMA is strik-
nificant effect on the cell length of P. avenae. The ing. In a few cases, DFMA was as effective or more
effect on mycelial growth of inhibitors of PA biosyn- effective than DFMO (Galston and Weinstein, 1988).
thesis is dependent upon the particular fungus (Rajam This could be due to the conversion of DFMA and
and Galston, 1985; Birecka et al., 1986; Khan and DFMO by arginase (Slocum and Galston, 1985; Slo-
Minocha, 1989; West and Walters, 1989; Foster and cum et al., 1988). DFMO has been reported to possess
Walters, 1990). This may be because of differences some systemic activity (Rajam et al., 1985), but very
between genera in the inhibitor uptake and distribu- little is known about its movement within the plant
tion within the cell, sensitivity of the corresponding and subsequent effects on fungal infection (Slocum,
enzymes or differences in the required cellular concen- 1991). Poor translocation of DFMO was observed in
tration of PAs for growth and morphogenesis (Birecka wheat plants (Slocum, 1991) suggesting that control of
et al., 1986; West and Walters, 1989; Foster and Wal- fungal pathogen through inhibition of PA biosynthesis
ters, 1990). Few patents have been granted in the alone is unlikely.
field of plant protection by the use of PA biosynthetic
inhibitors (Table 2).
Fungi are known only to use the ODC pathway Mutational analysis
for Put synthesis. Khan and Minocha (1989) have re-
ported that two phytopathogenic fungi (Ceratocystis In Petunia and tomato, floral-morphology mutants
minor and Verticillium dahliae) contain significant have been shown to possess increased put content
levels of ADC activity with very little ODC activ- and ADC activity; however the molecular analysis
ity. In these fungi ADC was shown to be inhibited of the mutants have not been carried out in detail
by α-DFMA, Put and Spd. The growth was strongly (Gerats et al., 1988; Rastogi and Sawhney, 1990).
inhibited by α-DFMA while α-DFMO had little effect. The mutants from mgBG and DFMO-resistant tobacco
Rajam et al. (1985) noticed that DFMO totally show an array of phenotypic characteristics including
inhibited rust uredospore germination on infected floral aberrations. Malmberg and Watson (1996) have
bean leaf surfaces, and this effect was partially re- identified a number of alleles defective in ADC in Ar-
versed with the application of Put or Spd to DFMO- abidopsis, which show minor alterations in root, stem,
treated leaves. There was a marked inhibition of and floral morphogenesis. The allele deficient in ODC
spore germination and of hydrolytic enzymes follow- activity is generally hypomorphs, with low but non-
ing DFMO and bis(cyclohexylammonium)sulphate zero levels of enzyme activity and the put content. In
(BCHA) treatments. another genetic approach, the SAMdc gene of potato
22
Table 2. Patents pertaining to polyamine biosynthesis inhibitors used for protection plant

Patent Title Description Inventor and

number applicant (year)

US 4376116 Polyamine biosynthesis Synthesis of derivatives of J.K. Coward & K.-C.

inhibitors. S-adenosyl-(1,8-diamino-3- Tang, The Dow
thiooctane) as polyamine Chemical Company,
biosynthesis inhibitors. USA (1983)

US 4760091 Method of controlling Application of fungicidally C.M. Carson &

phytopathogenic effective amount of P.P. McCann, British
fungus. ornithine decarboxylase Technology Group
inhibitor for controlling rust Ltd., UK (1988)
and smut fungi of order
Uredinales.

US 5461079 Antifungal compounds Use of (E)-2-butene-1,4- D.J. Robins & D.R.

-diamine or a salt thereof Walters, Research
with inorganic or organic Corporation, USA
acids, as a fungicide. (1995)

US 4751348 Nicotiana plants with Plant cell lines with altered R.L. Malmberg & J.I.
altered PA levels and flower structures and altered MacIndoo, Cold
flower structures. male/female sterility and Spring Harbor
altered PA biosynthesis Laboratory, NY, USA
have been isolated using UV (1988)
mutagenizing light, PA
biosynthetic inhibitors and
growth in dark.

US 4818770 Prevention of a plant α-DFMO an inhibitor of the L.H. Weinstein &

disease by specific PA biosynthetic enzyme A.W. Galston, Boyce
inhibition of fungal ODC strongly retards the Thomas Institute of
polyamine biosynthesis. growth of several species of Plant Research,
phytopathogenic fungi in Ithaca, NY, USA
vitro. Such inhibitions can (1989)
be completely reversed by
Put or Spd, confirming the
essentiality of PAs for
growth of fungal hyphae, this
use of α-DFMO can be
utilized in plant protection
against pathogens.

has been mapped to chromosome 5 and shows genetic detail, SAMdc activity was increased in callus, and in
linkage with the tuberization process. one line this was coupled with elevated levels of Put
Activation T-DNA tagging has been used to cre- and Spd. A mutant line of Arabidopsis thaliana carry-
ate MGBG-resistant tobacco cell lines (Fritze et al., ing an insertion of the En-1 transposable element at the
1995). The regenerated plants from MGBG-resistant ADC2 locus as been isolated. The insertion was found
lines display characteristic leaf and floral malforma- to cause a knock out of the ADC2 gene, which is re-
tion as well as parthenocarpy. In two lines studied in
 23
Table 3. Cloned genes of polyamine biosynthesis pathway (Modified from Kumar et al. 1997)

Gene Organism Reference

SAM synththase Arabidopsis Peleman et al., 1989

Dianthus caryophyllus Larsen & Woodson, 1991
Petroselinum hortense Kawalleck et al., 1992
Poplar deltoids×P. richocarpa Doorsselaere et al., 1993

ADC Tomato Espartero et al., 1994; Rastogi et al., 1993

Oat Bell & Malmberg, 1990
Arabidopsis Watson & Malmberg, 1996
Dianthus caryophyllus Chang et al., 1996
Aethionema grandiflora Galloway et al., 1998
Theobroma cacoa Galloway et al., 1998
Pea Perez-Amador et al., 1995

ODC Soybean Nam et al., 1996

Datura stramonium Michael et al., 1996; Taylor et al., 1992
Tobacco Hamill et al., 1990

SAMdc Tomato Alabadi & Carbonell, 1998; R. Fray (Kumar et al., 1997)
Potato Mad-Arif et al., 1994
Ipomoea nil Park et al., 1998
Vicia faba Freuhling et al., 2000
Spinach Bolle et al., 1995
Periwinkle Schroeder & Schroeder, 1995; Chang et al., 1996

Homospermidine synthase Tritorduem Dresselhaus et al., 1996

Tobacco S. Paramale (Kumar et al., 1997)
Carnation Schroeder & Schroeder, 1995
Senecio vulgaris Kaiser, 1999

sponsible for the induction of PA biosynthetic pathway its effect on plant developmental processes and stress
by osmotic stress (Soyka and Heyer, 1999). using transgenic and molecular genetic approaches.
Various PA biosynthetic enzyme genes have been
cloned and expressed in various plant systems (Table
Molecular understanding of PAs and their 3).
modulation in transgenics ADC, the first key enzyme in the altered pathway
plays a pivotal role in the biogenesis of precursor Put.
In recent years, ADC has been cloned from oat (Bell
Genes for several key biosynthetic enzymes like ADC,
and Malmberg, 1990), tomato (Rastogi et al., 1993),
ODC and SAMdc have been cloned from different
pea (Perez-Amador et al., 1995), arabidopsis (Watson
plant species (Chattopadhyay and Ghosh, 1998). The
and Malmberg, 1996) and soybean (Nam et al., 1996).
use of molecular approach including the cloning of
Each of the cloned ADC genes is similar to the bio-
genes for PA biosynthetic enzymes, production of
synthetic E. coli ADC and encodes proteins containing
transgenic plants, isolation and characterization of
the conserved, putative substrate-binding site that has
mutants defective in PA biosynthesis will provide a
been found in all eukaryotic ADCs and ODCs reported
better understanding of the role of PAs in higher
so far. This fact suggests a similar catalytic mechanism
plants. New impetus to PA biosynthetic research has
(Poulin et al., 1992).
come with the cloning of many PA biosynthetic genes
and the cellular perturbation of PA levels and studying
24

The cDNA clone representing ODC from Datura that the effects of genes encoded by the Ri-T-DNA
has been obtained by Michael et al. (1996). It is similar on flowering might be brought about through an in-
to other eukaryotic ODCs, but lacks long 3 and 5 un- hibitor of polyamine biosynthesis. Martin-Tanguy et
translated regions that are present in mammalian ODC al. (1990), hypothesized that the Ri-TDNA encodes
mRNAs and are probably involved in translational effi- genes which represses polyamine formation by alter-
ciency. A cDNA of tobacco BY-2 cells corresponding ing the activity of enzymes (ODC/ADC) encoded by
to an mRNA species, which was rapidly induced by plant genome, but the system specificity in each case
methyl jasmonate in the presence of cycloheximide, can not be ruled out for the modification of the activity
was found to encode ODC. Methyl jasmonate has been of genes. Martin-Tanguy et al. (1991) in the extension
shown to sequentially induce expression of a series of their previous work have proved cytogenetically, the
of genes involved in nicotine biosynthesis by multiple influence of PAs in morphological response in trans-
regulatory mechanisms (Imanishi et al., 1998). genic tobacco, wherein the inhibition of ADC/ODC
SAMdc has been observed in carrot (Montague by polyamine biosynthetic inhibitor DFMA/DFMO
et al., 1979), oat (Kaur-Sawhney et al., 1988) and resulted in negative morphogenetic response, in this
tobacco (Malmberg, 1983). Genes for SAMdc have report they found out that Ri-T-DNA expresses and
been cloned from potato (Mad-Arif et al., 1994), spin- change in polyamine metabolism, any one gene out of
ach (Bolle et al., 1995), periwinkle (Schroeder and 18 ORFs could have resulted in pleiotrophic response.
Schroeder, 1995), carnation (Chang et al., 1996) and They observed that rol C gene from A. rhizogenes
Tritordeum (Dresselhaus et al., 1996). There is a resulted in inhibition of floral morphogenesis, with
strong (70%) sequence similarity, at the level of de- a decrease in free and conjugated polyamine titers
duced amino acid sequences among the plant SAMdc (Martin-Tanguy et al., 1991). This effect of partial
genes, but they have only 30–35 and 20–26% se- hindrance in apical domain was restored to an extent
quence identity with the mammalian and bacterial by feeding of PAs. Thus rol C was correlated with
SAMdc (Atmar and Kuehn, 1981; Recsei and Snell, repression of decarboxylases (Martin-Tanguy et al.,
1984; Schroeder and Schroeder, 1995; Chang et al., 1991). But the rol A gene, which is under the regu-
1996; Dresselhaus et al., 1996). Potato SAMdc is lation of a wild promoter, and in turn influence the
highly expressed in actively dividing and differentiat- morphological alteration in the transgenic plant, which
ing tissues of both vegetative and reproductive organs includes reduced internodes, severe leaf wrinkling, in-
(Mad-Arif et al., 1994). hibition of flowering, and these deleterious effect of
Plant transformation mediated by A. tumefa- rol A cannot be reversed to that normal level by wa-
ciens/A. rhizogenes, effects several biochemical tering of PAs (Martin-Tanguy et al., 1991). Sun et al.
changes, evident by several lines of work (Quattro- (1991) have correlated the impaired morphological re-
chio et al., 1986; Shen et al., 1988; Spano et al., sponse in transgenic tobacco with respect to its lower
1988). Whole plants containing Ri/Ti plasmid T-DNA level of polyamine metabolism and phenotypic aber-
display a truncated phenotype that includes alteration rations to the expression of rol A gene. It is observed
in developmental processes including flowering, ap- that rol A phenotype is correlated with the inhibition
ical dominance and leaf morphology (Tepfer, 1982, of the formation of di-p-coumeroyl putrescine and di-
1984; Shen et al., 1988; Spano et al., 1988). This p-coumeroyl spermidine prior to anthesis, hence rol A
phenotype is sexually transmitted as a set of domin- inhibits the conjugate formation, leaving free PAs in-
ant traits that co-segregate with the transferred gen- tact and affecting conjugates to the maximum, thereby
otype (Tepfer, 1984). Martin-Tanguy et al. (1990) affecting the methyltransferases activity in polyamine
have shown that the presence of Ri-T-DNA in to- metabolism.
bacco transgenic plants, the transformed phenotype In our laboratory A. rhizogenes mediated trans-
showed alterations in PA metabolism and accumula- formation of chicory resulted in altered phenotypes,
tion. They have shown an inverse relationship between associated with an altered morphogenetic behaviour
the levels of phenotypic changes and polyamine levels with regard to shoot multiplication, in vitro flower-
and their conjugates, wherein they have postulated ing, altered polyamine and ethylene biosynthesis (Bais
that the appearance of conjugated PAs as markers for et al., 2000b). The transgenic chicory showed lower
flowering. This postulate strengthens the hypothesis levels of endogenous PAs as compared to the normal
that conjugates may play a role in the transition from plants. The inhibition of polyamine metabolism can
vegetative to floral meristem. They even suggested be correlated to the expression of rol A gene in the
 25

regenerated plants, which was further confirmed by Capell et al. (1998) have reported the overexpres-
the polyamine inhibitor experiments. But in contrast sion of the oat ADC c-DNA in transgenic rice, which
of earlier reports of Martin-Tanguy et al. (1990) and resulted in enhanced accumulation of Put in trans-
Sun et al. (1991). Our experiments found that restor- genic rice varieties. Very recently, Bassie et al. (2000)
ation of morphological response was achieved upon have shown that the promoter strength influences the
exogenous polyamine administration in both normal PA metabolism and morphogenetic capabilities of the
and transgenic plants. transgenic rice tissues expressing oat ADC cDNA.
The yeast ODC genes over-expressed in tobacco The second-generation transgenic tobacco plants
hairy root cultivar produced up to 3-fold increase in over expressing an oat ADC cDNA contained high
ODC activity and doubled the nicotine content (Hamill levels of oat ADC transcript relative to tobacco ADC,
et al., 1990). Most of the transformants appeared nor- possessed elevated ADC enzyme activity and accu-
mal, although the ones having highest ODC and Put mulate 10–20-fold more agmatine, but there was no
levels display stunted growth, wrinkled leaves, and increase in the levels of PAs. Overaccumulation of
flowers with reduced stems. The tobacco transgenic agmatine in transgenic plants did not affect morpho-
with the human SAMDC (Noh and Minocha, 1996) logical development (Burtin and Michael, 1997).
shows a 2–4-fold increase in spd levels. ADC gene of Localization of ADC enzyme in the chloroplast
oat has been over-expressed in tobacco under the con- (Galston et al., 1997) suggests a role of PAs in the
trol of an inducible promoter; the Tet-repressor system maintenance of photosynthetic activity during senes-
and this shows a significant change in the levels of cence responses induced due to osmotic stress. The
ADC transcript and activity, and the Put content upon use of transgenic techniques has already demonstrated
tetracycline-induction (Masgrau et al., 1997). In to- this interaction in case of tuber formation in potatoes
bacco root, a correlation exists between the expression (Pedros et al., 1999). In other processes, for example
of the rol genes from the TL-DNA (rol A, rol B, rol fruit ripening (Li et al., 1992), somatic embryogenesis
C) of A. rhizogenes and polyamine metabolism. The (Bastola and Minocha, 1995) stress induced senes-
higher polyamine contents found in roots transformed cence (Masgrau et al., 1997) and flowering (Masgrau
by rol A parallel with higher ODC and ADC activit- et al., 1997) could now be examined. On the other
ies as well as higher nicotine contents (Altabela et al., hand there is also evidence that flux of the Put pools
1995). can be driven to primary and secondary pathways by
It can be inferred that the molecular dissection of means of genetic manipulation (Hamill et al., 1990).
the Ri-T-DNA will provide tools for evaluating the im- Therefore, manipulation of PA biosynthetic pathway
portance of PAs in the diverse developmental process may deserve closer attention in the future from bio-
in which they are implicated. technological point of view.
In other reports dealing with the transgenic re-
search on modulation of polyamine metabolism,
Bastola and Minocha (1995) showed the increased Conclusions and future line of work
Put biosynthesis through incorporation of mouse ODC
cDNA in carrot cell cultures for increased somatic em- In this review attempt has been made to discuss on
bryogenesis. Hamill et al. (1990) have shown the over- the role of polyamine in all the stages of life cycle
expression of polyamine metabolism by incorporation of the plant. Several biotechnological utilities of role
of a yeast ODC gene in transformed root cultures of polyamine and their metabolic inhibitors have been
of Nicotiana rustica, which further lead to increased dealt in detail.
nicotine production. Andersen et al. (1998) showed There is a gap in information on the translocation
the interaction of polyamine metabolism in transgenic of free polyamines and their direct interaction with
cells of carrot expressing mouse ODC cDNA, wherein hormone. Does polyamine play a role in gene ex-
they inferred that the accumulation of Put in the pression? Does it act directly or indirectly? Does an
transgenic cells was due to its increased biosynthesis indirect effect is mediated through calcium involve-
through the mouse ODC pathway. The transgenic and ment or other signal transduction pathways? Can we
nontransgenic cells showed similar rates of conversion metabolically engineer the pathways for regulating
of arginine to put. The transgenic cells degraded put at morphogenesis or for other biotechnological utility
higher rates as compared to the nontransgenic cells. such as secondary metabolite production? Does the
bound polyamines also have definite role? Is there re-
26

lease of bound polyamines for utility at the site of its Altman A (1982) Retardation of radish leaf senescence by polyam-
action? If so what are the specific carrier molecules? ines. Physiol. Plant 54: 189–193
Altman A, Kaur-Sawhney R & Galston AW (1977) Stabilisation
Their characterization will further help in our under- of oat leaf protoplast through polyamine mediated inhibition of
standing of the role of polyamines during the ontogeny senescence. Plant Physiol. 60: 570–574
of the plant. This needs to be analyzed not only at or- An LZ & Wang (1997) Changes in polyamine contents and argin-
gan level but also at cellular, subcellular and molecular ine decarboxylase activity in wheat leaves exposed to ozone and
hydrogen-fluoride. J. Plant Physiol. 150: 184–187
levels. Andersen SE, Bastola DR & Minocha SC (1998) Metabolism of
Hence the research on polyamine metabolism is at polyamines in transgenic cells of carrot expressing a mouse
an interesting stage and if we address these questions ornithine decarboxylas cDNA. Plant Physiol. 116: 299–307
we will understand an array of functions of these rel- Antognoni F, Casali P, Pistocchi R & Bagni N (1994) Kinetics and
calcium-specificity of polyamine uptake in carrot protoplasts.
atively simple molecules, which play a marvelous role Amino acids. 6: 301–309
in various stages of growth, development and adapta- Apelbaum A (1986) Polyamine involvement in the development and
tion of plants to the environment. Still there are more ripening of avocado fruit. Acta Horticult. 179: 779–785
unsolved questions than answers! Apelbaum A, Burgoon AC, Anderson JD, Lieberman M, Ben-Arie
R & Mattoo AK (1981) Polyamines inhibit biosynthesis of ethyl-
ene in higher-plant tissue and fruit protoplasts. Plant Physiol. 68:
453–456
Apelbaum A, Vinkler C, Sfakiotakis E & Dilley DR (1984) In-
Acknowledgements
creased mitochondrial DNA and RNA polymerase activity in
ethylene-treated potato tubers. Plant Physiol. 76: 461–464
HPB acknowledges the Council of Scientific and In- Apelbaum A, Goldlust A & Icekson I (1985) Control by ethylene of
dustrial Research (CSIR) New Delhi, for providing arginine decarboxylase activity in pea seedlings and its implica-
tion for hormonal regulation of plant growth. Plant Physiol. 79:
Senior Research Fellowship. Authors are thankful to 635–640
Ms. G. Sudha, of the Department of Plant Cell Bi- Atmar V & Kuehn G (1981) Phosphorylation of ornithine de-
otechnology, CFTRI, Mysore and Dr. Jacob George, carboxylase by a polyamine dependent protein kinase. Proc.
of the Department of Plant Pathology and Microbio- Natl. Acad. Sci. USA 78: 5518–5522
Audiso S, Bagni S & Serafini-Fracassini D (1976) Polyamines dur-
logy, School of Environmental Sciences, University ing growth in vitro of Nicotiana glauca habituated tissue. Z.
of Natal, South Africa for their valuable and timely Pflanzenphysiol. 81: 226–233
help in cataloging references for this review. This Aurisano N, Bertani A, Mattana M & Reggiani R (1994) Absicisic
acid induced stress-like polyamine pattern in wheat seedlings,
work was supported by the grant from Department
and its reversal by potassium ions. Physiol. Plant 89: 687–692
of Biotechnology (DBT), Government of India, New Bagga S, Rochford J, Klaene Z, Kuehn GD & Phillips GC (1997)
Delhi. Putrescine aminopropyltransferase is responsible for biosyn-
thesis of spermidine, spermine and multiple uncommon polyam-
ines in osmotic stress-tolerant alfalfa. Plant Physiol. 114: 445–
454
References Bagni N, Calzoni GL & Spernaza A (1978) Polyamines as sole ni-
trogen sources for Helianthus tuberosus explants in vitro. New
Adams DO & Yang SF (1979) Ethylene biosynthesis identification Phytol. 80: 317–323
of 1-aminocyclopropane-1-carboxylic acid as an intermediate in Bagni N, Adamo P & Serafini-Fracassini D (1981) RNA, proteins
the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. and polyamines during tube growth in germinating apple pollen.
USA 76: 170–174 Plant Physiol. 68: 727–730
Agazio M de, Giardina MC & Gregor S (1988) Effects of exogenous Bais HP (2000) Studies on growth of hairy root cultures of Ci-
putrescine, spermidine, and spermine on K+ uptake and H+ ex- chorium intybus L. and production of phytochemicals. Ph.D
trusion through plasma membrane in maize root segments. Plant Thesis, University of Mysore, Mysore, India
Physiol. 87: 176–178 Bais HP, George J & Ravishankar GA (1999a) Influence of polyam-
Alabadi D & Carbonell J (1998) Expression of ornithine de- ines on growth of hairy root cultures of witloof chicory (Ci-
carboxylase is transiently increased by pollination, 2,4- chorium intybus L. cv. Lucknow local) and formation of cou-
dichlorophenoxyacetic acid, and gibberellic acid in tomato marins. J. Plant Growth Regul. 18: 33–37
ovaries. Plant Physiol. 118: 323–328 Bais HP, Sudha G & Ravishankar GA (1999b) Putrescine influences
Alabadi D, Aguero MS, Perez-Amador MA & Carbonell J (1996) growth and production of coumarins in hairy root cultures of Ci-
Arginase, arginine decarboxylase, ornithine decarboxylase and chorium intybus L. cv. Lucknow local (witloof chicory). J. Plant
polyamines in tomato ovaries. Changes in unpollinated ovaries Growth Regul. 18: 159–165
and parthenocarpic fruits induced by auxin or gibberellin. Plant Bais HP, Bhagyalakshmi N, Rajasekaran T & Ravishankar GA
Physiol. 112: 1237-1244 (2000a) Influence of polyamines on growth and production of
Altabella T, Angel E, Biondi S, Palazon J, Bagni N & Pinol MT secondary metabolites in hairy root cultures of Beta vulgaris and
(1995) Effect of rol genes from Agrobacterium rhizogenes on Tagetes patula. Acta Physiol. Plant 22: 151–158
polyamine metabolism in tobacco roots. Physiol. Plant 95: 479– Bais HP, Sudha G & Ravishankar GA (2000b) Putrescine and sil-
485 ver nitrate influences shoot multiplication, in vitro flowering and
 27

endogenous titres of polyamines in Cichorium intybus L. cv. putrescine and conjugated polyamines in Hyoscyamus muticus L.
Lucknow local. J. Plant Growth Regul. 19: 238–248 root cultures. Plant Cell Rep. 19:691–697
Bajaj S & Rajam MV (1995) Efficient plant regeneration from long Birecka H, Bionti AJ & McCann PP (1985) Activities of argin-
term cultures of rice by spermidine. Plant Cell Rep. 14: 717–720 ine and ornithine decarboxylases in various plant species. Plant
Bajaj S & Rajam MV (1996) Polyamine accumulation and near Physiol. 79: 515–519
loss of morphogenesis in long-term callus cultures of rice. Plant Birecka H, Garraway MO, Baumann RJ & McCann PP (1986) In-
Physiol. 112: 1343–1348 hibition of ornithine decarboxylase and growth of the fungus
Balasundaram D & Tyagi AK (1991) Polyamine-DNA nexus: Helminthosporium maydis. Plant Physiol. 80: 798–800
structural raminfications and biological implications. Mol. Cell Birecka et al. (1989) Effect of light period on polyamine metabolism
Biochem. 100: 129–140 in various plant species. J. Plant Physiol. 22: 141–149
Baraldi R, Bertazza G, Bregoli AM, Fasolo F, Rotondi A, Predieri Bolle C, Herrmann RG & Oelmunier (1995) A spinach cDNA with
S, Serafini-Fracassini D, Slovin JP & Cohen JD (1995) Auxins homology to S-adenosylmethionine decarboxylase. Plant Physiol
and polyamines in relation to differential in vitro root induction 107: 1461–1462
on microcuttings of two pear cultivars. J Plant Growth Regul. 11: Bonneau L, Berangenovat N, Monin J & Martin-Tanguy T (1995)
21–31 Stimulation of root and somatic embryo production in Euonymus
Bassie L, Noury M, Lepri O, Lahaye T, Christou P & Capell T europaeus L. by an inhibitor of polyamine biosynthesis. Plant
(2000) Promoter strength influences polyamine metabolism and Growth Regul. 16: 5–10
morphogenic capacity in transgenic rice tissues expressing oat Borrell A, Besford RT, Altabella T, Masgrau C & Tiburcio AF
ADC cDNA constitutively. Transgenic Res. 9: 33–42 (1996) Regulation of arginine decarboxylase by spermine in
Basso LC & Smith TA (1974) Effect of mineral deficiency on amine osmotically stressed oat leaves. Physiol. Plant 98: 105–110
formation in higher plants. Phytochemistry. 13: 875–883 Botha ML & Whitehead C (1992) The effect of polyamines on ethyl-
Bastola RD & Minocha SC (1995) Increased putrescine biosyn- ene synthesis during normal and pollination-induced senescence
thesis through transfer of mouse ornithine decarboxylase cDNA of Petunia hybrida L. flowers. Planta. 188: 478–483
in carrot promotes somatic embryogenesis. Plant Physiol. 109: Burtin D & Michael AJ (1997) Overexpression of arginine de-
63–71 carboxylase in transgenic plants. Biochem. J. 325: 331–337
Baxter C & Coscia CJ (1973) In vitro synthesis of spermidine in Bush DS (1995) Calcium regulation in plant cells and its role in
higher plant Vinca rosea. Biochim. Biophys. Res. Commun. 54: signaling. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 95–122
147–154 Cabanne F, Dalebroux MA, Martin-Tanguy J & Martin C (1981)
Bell E & Malmberg RL (1990) Analysis of cDNA encoding arginine Hydroxy cinnamic-acid amides and ripening to flower of Nico-
decarboxylase from oat reveals similarity to the Escherichia coli tiana tabacum cultivar Xanthi N.C. Physiol. Plant 53: 399–404
arginine decarboxylase and evidence of protein processing. Mol. Capell T, Escobar C, Liu H, Burtin D, Lepri O & Christou P (1998)
Gen. Genet. 224: 431–436 Overexpression of the oat arginine decarboxylase cDNA in trans-
Belles JM, Perez-Amador MA, Carbonell J & Conejero V (1993) genic rice (Oryza sativa L.) affects normal development patterns
Correlation between ornithine decarboxylase and putrescine in in vitro and results in putrescine accumulation in transgenic
tomato plants infected by citrus-exocortis viroid or treated with plants. Theor. Appl. Genet. 97: 246–254
ethephon. Plant Physiol. 102: 933–937 Carbonell J & Navarro JL (1989) Ribulose-1,5-bisphosphate-
Ben-Arie R, Lurie S & Mattoo AK (1982) Temperature-dependent carboxylase and fruit set or degeneration of unpollinated ovaries
inhibitory effects of calcium and spermine on ethylene bio- of Pisum sativum. Planta 178: 482–487
synthesis in apple discs correlate with changes in microsomal Carson CM & McCann PP (1988) Method of controlling phytopath-
membrane microviscosity. Plant Sci. Lett. 24: 239–247 ogenic fungus. US Patent 4760091
Benavides MP, Aizencang G & Tomaro ML (1997) Polyamines in Chang KS, Nam KH, Lee MM, Lee SH & Park KY (1996) Nuc-
Helianthus annus L. during germination under salt stress. J. Plant leotide sequence of cDNA (accession number U63832) encod-
Growth Regul. 16: 205–211 ing S-adenosylmethionine decarboxylase from carnation flowers
Berlin J & Forche E (1981) DL-α-difluoromethylornithine causes (PGR 96-092). Plant Physiol 112: 863
enlargement of cultured tobacco cells. Z. Pflanzenphysiol. 101: Chattopadhyay MK & Ghosh B (1998) Molecular analysis of
272–282 polyamine biosynthesis in higher plants. Current Sci. 74: 517–
Bernal-Lugo I (1983) Relationship of polyamines, endogenous con- 522
tent and GA3 effects in barley aleurone layer. Plant Physiol. Chattopadhyay MK, Gupta SR, Sengupta DW & Ghosh B (1997)
72(Suppl.): 83(Abstr. 473) Expression of arginine decarboxylase in seedling of indica rice
Berta G, Altamura MM, Fusconi A, Cerruti F, Capitani F & Bagni (Oryza sativa L.) cultivars as affected by salinity stress. Plant
N (1997) The plant cell wall is altered by inhibition of polyamine Mol. Biol. 34: 477–483
biosynthesis. New Phytol. 137: 569–577 Cheng SH, Shyr YY & Kao CH (1984) Senescence in rice leaves.
Beyer EM Jr (1976) A potent inhibitor of ethylene action in plants. XII. Effect of 1,3-diaminopropane, spermidine and spermine.
Plant Physiol. 58: 268–271 Bot. Bull. Acad. Sin. 25: 191–196
Biasi R, Bagni N & Cista G (1988) Endogenous polyamines in Chi GL & Pua EC (1989) Ethylene inhibitors enhanced de novo
apples and their relationship in fruit set and fruit growth. Plant shoot regeneration from cotyledons of Brassica campestris spp.
Physiol. 73: 201–205 Chinensis (Chinese cabbage) in vitro. Plant Sci. 64: 243–250
Biasi R, Costa G & Bagni N (1991) Polyamine metabolism is related Chi GL, Barfield DG, Sim GE & Pua EC (1990) Effect of AgNO3
to fruit set and growth. Plant Physiol. Biochem. 29: 497–506 and aminoethoxyvinylglycine on in vitro shoot and root or-
Biddington NL (1992) The influence of ethylene in plant tissue ganogenesis from seedling explants of recalcitrant Brassica
culture. Plant Growth Regul. 11: 173–178 genotypes. Plant Cell Rep. 9: 195–198
Biondi S, Fornale S, Oksman-Caldentey KM, Eeva M, Agostani S & Chi GL, Pua EC & Goh CJ (1991) Role of ethylene on de novo shoot
Bagni N (2000) Jasmonates induce over-accumulation of methyl- regeneration from cotyledonary explants of Brassica campestris
28

spp. Pekinensis (Lour) Olsson in vitro. Plant Physiol. 96: 178– Dumortier FM, Flores HE, Shekhawat NS & Galston AW (1983)
183 Gradients of polyamines and their biosynthetic enzymes in
Chi GL, Lin WS, Lee JEE & Pua EC (1994) Role of polyamines coleoptiles and roots of corn. Plant Physiol. 72: 915–918
on de novo shoot morphogenesis from cotyledons of Brassica Egea-Cortines M & Mizrahi Y (1991) Polyamines in cell division,
campestris spp. Pekinensis (Lour) Olsson in vitro. Plant Cell fruit set and development, seed germination. In: Slocum RD &
Rep. 13: 323–329 Flores HE (eds) Biochemistry and Physiology of Polyamines in
Cho SC (1983) Effect of cytokinin and several inorganic cations on Plants. (pp. 143–158) CRC Press, Boca Raton, FL
the polyamine content of lettuce cotyledons. Plant Cell Physiol. Espartero J, Pintor-Toro JA & Pardo JM (1994) Differential ac-
24: 27–32 cumulation of S-adenosyl-methionine synthetase transcripts in
Chriqui D, D’Orazi D & Bagni N (1986) Ornithine and arginine response to salt stress. Plant Mol. Biol. 25: 217–227
decarboxylases and polyamine involvement during in vivo dif- Evans PT & Malmberg RL (1989) Do polyamines have a role in
ferentiation and in vitro dedifferentiation of Datura innoxia leaf plant development? Annu. Rev. Plant Physiol. Plant Mol. Biol.
explant. Physiol. Plant 68: 589–596 40: 235–269
Cohen E & Kende H (1986) The effect of submergence ethylene Even-Chen Z, Mattoo AK & Goren R (1982) Inhibition of ethyl-
and gibberellin on polyamines and their biosynthetic enzymes in ene biosynthesis by aminoethoxyvinylglycine and by polyamines
deepwater-rice internodes. Planta 169: 498–504 shunt label from C14-methionine into spermidine in aged orange
Cohen E, Arad SM, Heimer YM & Mizrahi Y (1982) Particip- peel discs. Plant Physiol 69: 385–388
ation of ornithine dcarboxylase in early stages of tomato fruit Feirer RP, Mignon G & Litvay JD (1984) Arginine decarboxylase
development. Plant Physiol. 70: 540–543 and polyamines required for embryogenesis in wild carrot. Sci-
Costa G & Bagni N (1983) Effects of polyamines on fruit-set of ence 223: 1433–1435
apple. Hortscience. 18: 59–61 Felix H & Harr J (1985) Association of polyamines to different parts
Coward JK & Tang KC (1983) Polyamine bisynthesis inhibitors. US of various plant species. Physiol. Plant 71: 245–250
Patent 4376116 Feray A, Hourmant A, Brun A & Penot M (1993) Effect of
Dai YR & Galston AW (1981) Simultaneous phytochrome- polyamines on morphogenesis of in vitro potato plants (Solanum
controlled promotion and inhibition of arginine decarboxylase tuberosum cv Bintje). CR Acad. Sci. Ser III 316: 1446–1451
activity in buds and epicotyls of etiolated pea. Plant Physiol. 67: Fienberg AA, Choi JH, Lubich WP & Sung ZR (1984) Devel-
266–269 opmental regulation of polyamine metabolism in growth and
Dai YR, Kaur-Sawhney R & Galston AW (1982) Promotion by differentiation of carrot culture. Planta 162: 532–539
gibberellic acid of polyamine biosynthesis in internodes of light- Flink L & Pettijohn DE (1975) Polyamines stabilize DNA folds.
grown dwarf peas. Plant Physiol. 69: 103–106 Nature 253: 62–63
Das S, Bose A & Ghosh B (1995) Effect of salt stress on polyam- Flores HE (1983) Studies on the physiology and biochemistry of
ine metabolism in Brassica campestris. Phytochemistry. 39: polyamines in higher plants, Ph.D Dissertation, Yale University.
283–285 Flores HE & Filner P (1984) Putrescine catabolism in plant cells:
Davies PJ (1987) The plant hormones: their nature, occurrence, and γ -aminobutyraldehyde dehydrogenase activity in mono and di-
functions. In: Davies PJ (ed), Plant Hormones and Their role cotyledonous species. Plant Physiol. 75(Suppl.): 118(Abstr.)670
in Plant Growth and Development, Ch. Al. Boston: Martinus Flores HE & Galston AW (1984a) Osmotic stress-induced polyam-
Nijhoff ine accumulation in cereal leaves. I. Physiological parameters of
Davis DG (1997) Polyamines, auxins and organogenesis in leafy the response. Plant Physiol. 75: 102–109
spurge (Euphorbia esula L.). J. Plant Physiol. 151: 603–609 Flores HE & Galston AW (1984b) Osmotic stress-induced polyam-
Davis DG & Olson PA (1994) Effects of putrescine and inhibitors ine accumulation in cereal leaves. II. Relation to amino acid pool.
of putrescine biosynthesis on organogenesis in Euphorbia esula Plant Physiol. 75: 110–113
L. In Vitro Cell. Dev. Biol. Plant 30P: 124–130 Floris G, Giartosio A & Rinaldi A (1983) Diamine oxidase from
Demeulemeester MAC & De Proft MP (1999) In vivo and in vitro Lens esculenta seedlings: Purification and properties. Phyto-
flowering response of chicory (Cichorium intybus L.): influence chemistry 22: 1871–1974
of plant age and vernalization. Plant Cell Rep. 18: 781–785 Foster SA & Walters DR (1990) The effects of polyamine
Dibble ARG, Davies PJ & Mutschler MA (1988) Polyamine content biosynthesis inhibitors on mycelial growth, enzyme activity
of long keeping alcoba tomato fruit. Plant Physiol. 86: 338–340 and polyamine levels in the oat-infecting fungus Pyrenophora
Doorssalaere JV et al. (1993) A cDNA encoding S-adenosyl- arenae. J. Gen. Microbiol. 136: 233–239
methionine synthetase from poplar. Plant Physiol 102: 1365– Friedman R, Altman A & Bachrach U (1982) Polyamines and root
1366 formation in mung bean hypocotyls cuttings. Plant Physiol. 70:
Downs CG & Lovell PH (1986) The effect of spermidine and pu- 844–848
trescine on the senescence of cut carnations. Physiol. Plant 66: Friedman R, Altman A & Bachrach U (1985) Polyamines and root
679–684 formation in mung bean hypocotyls cuttings. II. Incorporation of
Dresselhaus T, Barcelo P, Hagel C, Lorz H & Humbeck K (1996) precursors into polyamines. Plant Physiol. 79: 80–83
Isolation and characterization of a Tritordeum cDNA encod- Fritze K, Czaja I & Walden R (1995) T-DNA tagging of genes
ing S-adenosylmethionine decarboxylase that is circardian-clock influencing polyamine metabolism: isolation of mutant plant
regulated. Plant Mol. Biol. 30: 1021–1033 lines and rescue of DNA promoting growth in the presence of
Drolet G, Dumbroff EB, Legge RL & Thompson JE (1986) Radical a polyamine biosynthetic inhibitor. Planta 17: 261–271
scavenging properties of polyamines. Phytochemistry. 25: 367– Freuhling M, Puehler A & Perlick AM (2000) Isolation and char-
371 acterization of a full length cDNA (Accession No. AJ 250026)
Dumas E, Perdrizet E & Vallee J (1981) Evolution quantitative des encoding S-adenosylmethionine decarboxylase from broad bean
acides amines et amines libres au cours du developpement de (Vicia faba L.). Plant Physiol. 122: 620
diverses especes de Nicotiana. Physiol. Veg. 19: 155–165
 29

Fuhrer J, Kaur-Sawhney R, Shih LM & Galston AW (1982) Effects Guranowski AB, Chiang PK & Cantoni GL (1981) 5 -
of exogenous 1,3-diaminopropane and spermidine on senescence Methylthioadenosine nucleosidase, purification and
of oat leaves. Plant Physiol. 70: 1597–1600 characteristics of the enzyme from Lupinus luteus seeds.
Gallardo M, Bueno M, Angosto T, Gallardo E & Mattilla AG (1992) Eur. J. Biochem. 114: 293–299
Free polyamines in Cicer arietinum seeds during the onset of Hamill JD, Robins RJ, Parr AJ, Evans DM, Furze JM & Rhodes
germination. Phytochemistry 31: 2283–2287 MJC (1990) Over-expression of a yeast ornithine decarboxylase
Galloway GL, Malmberg RL & Price RA (1998) Phylogenetic util- gene in roots of transgenic Nicotiana rustica can lead to en-
ity of the nuclear gene ADC: an example from Brassicaceae. hanced nicotine accumulation. Plant Mol. Biol. 15: 27–38
Mol. Biol. Evol. 15: 1312–1320 Hamana K & Matsuzaki S (1982) Widespread occurrence of nor-
Galston AW & Kaur-Sawhney R (1987) Polyamines as endogenous spermidine and norspermine in eukaryotic algae. J. Biochem. 91:
growth regulators. In: Davies PJ (eds) Plant Hormones and their 1321–1328
role in Plant Growth and Development. pp. 280–295. Martinus Havelange A, Lejeune P, Bernier G, Kaur-Sawhney R & Galston
Nijhoff, Dordrecht AW (1996) Putrescine export from leaves in relation to floral
Galston AW (1983) Polyamines as modulators of plant develop- transition in Sinapis alba. Physiol. Plant 96: 59–65
ment. Bioscience 33: 382–388 Hayyim GB, Damon JP, Martin-Tanguy J & Tepfer D (1994) Chan-
Galston AW & Weinstein LH (1988) Control of phytopathogens by ging root system architecture through inhibition of putrescine
inhibitors of polyamine biosynthesis. Adv. Exp. Med. Biol. 250: and feruloyputrescine accumulation. FEBS Lett. 342: 145–148
589–599 Heby O (1981) Role of polyamines in the control of cell prolifera-
Galston AW, Altman A & Kaur-Sawhney R (1978) Polyamines, tion and differentiation. Differentiation 19: 1–20
ribonuclease, and the improvement of oat leaf protoplasts. Plant Heimer YM & Mizrahi Y (1982) Characterization of ornithine de-
Sci. Lett. 11: 69–79 carboxylase of tobacco cells and tomato ovaries. Biochem. J.
Galston AW, Kaur Sawhney R, Altabella T & Tiburcio AF (1997) 201: 373–376
Polyamines in reproductive activity and response to abiotic Heimer YH, Mizrahi Y & Bachrach U (1979) Ornithine de-
stress. Bot Acta. 110: 197–207 carboxylase activity in rapidly proliferating plant cells. FEBS
Gamarink A, Frydman RB & Barreto D (1994) Prevention of infec- Lett. 104: 146–149
tion of soybean seeds by Colletotrichum truncatum by polymine Helleboid S, Couillerot JP, Hilbert JH & Vasseur J (1995) Inhibition
biosynthesis inhibitors. Phytopathology. 84: 1445–1448 of direct somatic embryogenesis by α-difluoromethylarginine in
Garcia-Espana A, Carbonell J & Rubio V (1989) Carbamoyl phos- a Cichorium hybrid: effects on polyamine content and growth
phate synthetase, ornithine transcarbamylase and aspartate tran- patterns. Planta 196: 571–576
scarbamylase activities in the pea ovary. Changes with senes- Hiatt AC & Malmberg RL (1988) Utilization of putrescine in to-
cence of the unpollinated ovary or with fruit set induced by bacco cell lines resistant to inhibitors of polyamine synthesis.
gibberellic acid. Plant Physiol. 90: 1565–1569 Plant Physiol. 86: 441–446
Garrido RL, Chibi F & Matila A (1995) Polyamines in the induc- Hibi N, Fujita T, Hatano M, Hashimoto T & Yamada Y (1992)
tion of Nicotiana tabacum pollen embryogenesis by starvation. J Putrescine-N-methyl transferase in cultured roots of Hyocyamus
Plant Physiol 145: 731–735 albus. n-Butylamine as a potent inhibitor of the transferase both
Gerats AGM, Kaye C, Collins C & Malmberg RL (1988) Polyam- in vitro and in vivo. Plant Physiol. 100: 826–835
ine levels in Petunia genotypes with normal and abnormal floral Hirasawa E & Suzuki Y (1983) Biosynthesis of spermidine in maize
morphologies. Plant Physiol 86: 390–393 seedlings. Phytochemistry. 22: 103–106
Giovanelli J, Mudd SH & Datko AH (1981) Recycling of me- Icekson I, Goldlust A & Apelbaum A (1985) Influence of ethylene
thionine sulfur in a higher plant by two pathways characterized on S-adenosyl methionine decarboxylase activity in etiolated pea
by either loss or retention of the 4-carbon moiety. Biochim. seedlings. J. Plant Physiol. 119: 335–345
Biophys. Acta 100: 831–839 Imanishi S, Hashizume K, Nakakita M, Kojima H, Matsubayashi
Gonzalez-Aguilar GA, Gayosso L, Cruz R, Fortiz J, Bacz R & Y, Hashimoto T, Sakagami Y, Yamada Y & Nakamura K (1998)
Wang CY (2000) Polyamines induced by hot water treatments Differential induction by methyl jasmonate of genes encoding
reduce chilling injury and decay in peppre fruit. Postharvest Biol. ornithine decarboxylase and other enzymes involved in nicotine
Technol. 18: 19–26 biosynthesis in tobacco cell cultures. Plant Mol. Biol. 38: 1101–
Goren R, Palavan N, Flores HE & Galston AW (1982a) Changes 1111
in polyamine titer in etiolated pea-seedlings following red-light Jarvis BC, Yasmin S & Coleman MT (1985) RNA and protein meta-
treatment. Plant Cell Physiol. 23: 19–26 bolism during adventitious root formation in stem cuttings of
Goren R, Palavan N & Galston AW (1982b) Separating phyto- Phaseolus aureus cultivar berkin. Physiol. Plant 64: 53–59
chrome effects on arginine decarboxylase activity from its effects Jirage DB, Ravishankar GA, Suvarnalatha G & Venkataraman LV
on growth. J Plant Growth Regul. 1: 61–73 (1994) Profile of polyamines during sprouting and growth of
Greenland AJ & Lewis DH (1984) Amines in barley leaves infected saffron (Crocus sativus L.) corms. J. Plant Growth Regul. 13:
by brown rust and their possible relevance to formation of ‘green 69–72
islands’. New Phytol. 96: 283–291 Kaiser A (1999) Cloning and expression of a cDNA encoding ho-
Grimes HD, Slocum RD & Boss WF (1986) α- mospermidine synthase from Senecio vulgaris (Asteraceae) in E.
Difluoromethylarginine treatment inhibits protoplast fusion coli. Plant J. 19: 195–201
in fusogenic carrot protoplasts. Biochim. Biophys. Acta 886: Kakkar RK & Rai VR (1987) Effects of spermine and IAA on carbo-
130–134 hydrate metabolism during rhizogenesis in Phaseolus vulgaris.
Guergue A, Claparols I, Santos M & Torne JM (1997) Modulator 1. Hypocotyl cuttings. Indian J Exp. Bot. 25: 476–478
effect of DL-α-difluoromethylarginine treatments on differenti- Kallio A, McCann P & Bey P (1981) DL-α-(difluoro-
ation processes of young maize calluses. Plant Growth Regul. methyl)arginine: a potent enzyme-activated inhibitor of
21: 7–14 bacterial arginine decarboxylase. Biochemistry. 20: 3163–3166
30

Kaur-Sawhney R & Galston AW (1979) Interaction of polyamines Lee TM (1997) Polyamine regulation of growth and chilling toler-
and light on biochemical processes involved in leaf senescence. ance of rice (Oryza sativa L.) roots cultured in vitro. Plant Sci.
Plant Cell Environ. 2: 189–196 122: 111–117
Kaur-Sawhney R, Altman A & Galston AW (1978) Dual mechanism Lee TM & Lin YH (1996) Opposite effects of Fusicoccin and IAA
in polyamine mediated control of ribonuclease activity in oat leaf on putrescine synthesis of rice coleoptiles. Physiol. Plant 97: 63–
protoplasts. Plant Physiol. 62: 158–160 68
Kaur-Sawhney R, Flores HE & Galston AW (1981) Polyamine ox- Lee TM, Lur HS, Lin YH & Chu C (1996) Physiological and bio-
idase in oat leaves: a cell wall localized enzyme. Plant Physiol. chemical related to methyl jasmonate-induced chilling tolerance
68: 494–498 of rice (Oryza sativa L.) seedlings. Plant Cell Environ. 19: 65–74
Kaur-Sawhney R, Shih LM & Galston AW (1982) Relation of Li N, Parsons BL, Liu DR & Mattoo AK (1992) Accumulation of
polyamine biosynthesis in the initiation of sprouting in potato wound-inducible ACC synthase transcripts in tomato fruit is in-
tubers. Plant Physiol. 69: 411–415 hibited by salicylic acid and polyamines. Plant Mol. Biol. 18:
Kaur-Sawhney R, Tiburcio AF & Galston AW (1988) Spermidine 477–487
and floral bud differentiation in thin layer explants of tobacco. Lieberman N (1979) Biosynthesis and action of ethylene. Annu.
Planta 173: 282–284 Rev. Plant Physiol. 30: 533–591
Kawalleck P, Plesch G, Hahlbrock K & Somssich IE (1992) Induc- Lin PPC (1984) Polyamine metabolism and its relation to response
tion by fungal elicitor of S-adenosyl-methionine synthetase and of the aleurone layers of barley seeds to gibberellic acid. Plant
S-adenosyl-L-homocysteine hydrolase mRNA in cultured cells Physiol. 74: 975–983
and leaves of Petroselinum crispum. Proc. Natl. Acad. Sci. USA Liyama K, Bach-Tuyet, Lam T & Stone BA (1994) Covalent cross
89: 4713–4717 links in the cell wall. Plant Physiol. 104: 315–320
Ke D & Romani RJ (1988) Effects of spermidine on ethylene pro- Mad-Arif SA, Taylor MA, George LA, Sutler AR, Burch LC, Dav-
duction and the senescence of suspension-cultured pear fruit ies HV, Stark MJR & Kumar A (1994) Characterisation of the
cells. Plant Physiol. Biochem. 26: 109–116 S-adenosylmethionine decarboxylase (SAMDC) gene of potato.
Khan MB & Harborne JB (1991) Potassium deficiency increases Plant Mol. Biol. 26: 327–338
tropane alkaloid synthesis in Atropa acuminata via arginine and Mader JC (1997) Studies on polyamines in Solanum tuberosum in
ornithine decarboxylase levels. Phytochemistry 30: 3559–3563 vitro. Effects of DFMO, DFMA, chlorogenic acid and putrescine
Khan MB & Minocha SC (1989) Biosynthetic ADC in phytopatho- on the endogenous distribution of polyamines, tuberisation and
genic fungi. Life Sci. 44: 1215–1222 morphology. J. Plant Physiol. 150: 141–152
Khan MB & Minocha SC (1991) Polyamines and somatic em- Mader JC & Hanke DE (1997) Polyamine sparing may be involved
bryogenesis in carrot II: the effects of CHA phosphate. J Plant in the prolongation of cell division due to inhibition of phenyl-
Physiol. 137: 446–452 propanoid synthesis in cytokinin-starved soybean cells. J. Plant
Khurana N, Saxena RK, Gupta R & Rajam MV (1996) Polyamines Growth Regul. 16: 89–93
as modulators of microcycle conidiation in Aspergillus flavus. Malmberg RL (1980) Biochemical, cellular, and developmental
Microbiology 142: 517–523 characterization of a temperature sensitive mutant of Nicotiana
Klein H, Priebe A & Jager HJ (1979) Putrescine and spermidine in tabacum and its second site revertant. Cell 22: 603–629
peas: effects of nitrogen source and supply. Physiol. Plant 45: Malmberg RL (1983) Mutants of Nicotiana tabacum that alter
497–499 polyamine synthesis. In: Randall DD (ed.) Current Topics in
Koenig H, Goldstone A & Lu CY (1983) Polyamines regulate cal- Plant Biochemistry and Physiology, 2: 211–221. Univ. Missouri
cium fluxes in a rapid plasma membrane response. Nature 305: Press, Columbia
530–534 Malmberg RL & McIndoo (1988) Nicotiana plants with altered
Kong L, Atree SM & Fowke LC (1998) Effects of PEG and MGBG polyamine levels and flower structure. US Patent 4751348
on endogenous levels and somatic embryo maturation in white Malmberg RL & Watson MB (1996) Genetic analysis of polyamine
spruce. Plant Sci. 133: 211–220 biosynthesis in Arabidopsis (abstract no. 41001). Plant Physiol.
Kumar A, Altabella T, Taylor MA & Tiburcio AF (1997) Recent 111: S-21
advances in polyamine research. Trends Plant Sci. 2: 124–130 Mansour MMF & Al-Mutawa MM (1999) Stabilization of plasma
Kushad MM, Richardson DG & Ferro AJ (1982) 5 - membrane by polyamines against salt stress. Cytobios 100: 7–17
Methylthioribose kinase activity in plants. Biochim. Biophys. Martin-Tanguy J, Perdrizet E & Martin C (1982) Hydroxycinnamic
Res. Commun. 108: 167–173 acid amides in fertile and cytoplasmic male sterile lines of maize.
Kushad MM, Richardson DG & Ferro AJ (1983) Intermediates in Phytochemistry 21: 1939–1945
the recycling of 5’-methylthioribose to methionine in fruits. Plant Martin-Tanguy J, Tepfer D, Paynot M, Burtin D, Heisler L & Martin
Physiol. 73: 257–261 C (1990) Inverse relationship between polyamine levels and the
Kushad MM, Yelenoski G & Knight R (1988) Interrelationship degree of phenotypic alteration induced by the root-inducing left-
of polyamine and ethylene biosynthesis during avocado fruit hand transferred DNA from Agrobacterium rhizogenes. Plant
development and ripening. Plant Physiol. 87: 463–467 Physiol. 92: 912–918
Kyriakidis DA (1983) Effect of plant growth hormones and polyam- Martin-Tanguy J, Tepfer D & Burtin C (1991) Effects of Ri TL-
ines on ornithine decarboxylase activity during the germination DNA from Agrobacterium rhizogenes and the inhibitors of
of barley seeds. Physiol. Plant 57: 499–504 polyamine synthesis on growth, floral development, sexual or-
Larsen RB & Woodson WR (1991) Cloning and nucleotide se- ganogenesis and polyamine metabolism in tobacco. Plant Sci. 80:
quence of an S-adenosyl-methionine synthetase cDNA from 131–144
carnation. Plant Physiol. 96: 997–999 Masgrau C, Altabella T, Farrais R, Besford RD, Tompson A & Ti-
Laukkanen H & Sarjala T (1997) Effect of exogenous polyamines burcio AF (1997) Inducible expression of oat ADC in transgenic
on scots pine callus in vitro. J. Plant Physiol. 150: 167–172 tobacco plants. Plant J. 11: 465–473
Lee T & Chu C (1992) Ethylene-induced polyamine accumulation
in rice coleoptiles. Plant Physiol. 100: 238–245
 31

Mattoo AK, Baker JE, Chalutz E & Lieberman M (1977) Effect Oshima T, Hamasaki N & Uzawa T (1988) Biochemical proper-
of temperature on the ethylene-synthesising systems in apple, ties of unusual polyamines found in an extreme thermophile;
tomato and Penicillum. Plant Cell Physiol. 18: 715–719 Thermus thermophilus. Adv. Exp. Med. Biol. 250: 633–642
McCann PP, Tardif C & Mamont PS (1987) Regulation of ornith- Pajula RL & Raina A (1979) Methylthioadenosine, a potent inhib-
ine decarboxylase by ODC antizyme in HTC cells. Biochem. itor of spermine synhtetase from bovine brain. FEBS Lett. 99:
Biophys. Res. Commun. 75: 948–954 343–345
Meijer EGM & Simmonds J (1988) Polyamine levels in rela- Palavan N & Galston AW (1982) Polyamine biosynthesis and
tion to growth and somatic embryogenesis in tissue cultures of titer during various developmental stages of Phaseolus vulgaris.
Medicago sativa L. J. Exp. Bot. 203: 787–794 Physiol. Plant 55: 438–444
Mengoli M, Chriqui D & Bagni N (1992) Putrescine biosynthesis Palavan N, Goren R & Galston AW (1984) Effects of some growth
and oxidation in normal and hairy root tobacco plants. J. Plant regulators on polyamine biosynthetic enzymes in etiolated pea
Physiol. 140: 153–155 seedlings. Plant Cell Physiol. 25: 541–546
Messiaen J & Van Cutsem P (1999) Polyamines and pectins. II. Palavan-Unsal N (1987) Polyamine metabolism in the roots of
Modulation of pectic-signal transduction. Planta 208: 247–256 Phaseolus vulgaris-interaction of the inhibitors of polyamine
Messiaen J, Cambier P & Van Cutsem P (1997) Polyamines and biosynthesis with putrescine in growth and polyamine biosyn-
pectins. I. Ion exchange and selectivity. Plant Physiol. 113: 387– thesis. Plant Cell Physiol. 28: 565–572
395 Palfreyman MG, McCann PP, Lovenberg W, Temple JG Jr & Sjo-
Metcalf BW, Bey P, Dauzin C, Jung MJ, Casava P & Ververt JP erdsma A (eds) (1989) Enzymes as Targets for Drug Design,
(1978) Catalytic irreversible inhibition of mammalian orniltrine Academic Press, New York
decarboxylase by substrate and product analogues. J. Amer. Palmer EE (1992) Enhanced shoot regeneration from Brassica
Chem. Soc. 100: 2551–2553 campestris by silver nitrate. Plant Cell Rep. 11: 541–545
Michael AJ, Furze JM, Rhodes MJC & Burtin D (1996) Molecu- Park WK, Lee SH & Park KY (1998) Cloning and characteriz-
lar cloning and functional identification of a plant ornithine ation of genome clone (Accession No. U64927) encoding S-
decarboxylase cDNA. Biochem. J. 314: 241–248 adenosylmethionine decarboxylase whose gene expression was
Minocha R, Kvaalen H, Minocha SC & Long S (1993) Polyamines regulated by light in morning glory (Ipomoea nil). Plant Physiol.
in embryogenic cultures of Norway spruce and red spruce. Tree 116: 867
Physiol. 13: 365–377 Patil P, Chandra R, Sangeeta K & Raghuveer P (1999) Influence of
Minocha SC & Minocha R (1995) Role of polyamines in somatic polyamines and ethylene inhibitors on somatic embryo induction
embryogenesis. In: Bajaj TPS (ed.) Biotechnology in Agricul- in chickpea (Cicer arietinum L.). Ind. J. Plant Physiol. 3: 26–31
ture and Forestry, Vol. 30; Somatic Embryogenesis and Synthetic Pedros AR, Macleod MR, Ross HA, McRae D, Tiburcio AF, Davies
Seeds (pp. 53–70). Springer, Berlin HV & Taylor MA (1990) Manipulation of SAMdc activity in
Miyazaki JH & Yang SF (1987) The methionine salvage pathway in potato tubers, an increase in activity leads to an increase in tuber
relation to ethylene and polyamine biosynthesis. Physiol. Plant number and change in tuber size. Planta 209: 153–160
69: 366–370 Pedroso MC, Primikiros N, Roubelakis A & Pais MA (1997)
Mizrahi Y & Heimer YM (1982) Increased activity of ornithine Free and conjugated polyamines in embryogenic and non-
dcarboxylase in tomato ovaries induced by auxin. Physiol. Plant embryogenic leaf regions of Camellia leaves before and during
54: 367–368 direct somatic embryogenesis. Plant Physiol. 101: 213–217
Montague MJ, Armstrong TA & Jarworski EG (1979) Polyamine Pegg AE (1983) In: Tabor H & Tabor CW (eds), Methods in
metabolism in embryogenic cells of Daucus carota. II. Changes Enzymology, Vol 94 (pp. 239–247) Academic Press, New York
in arginine decarboxylase activity. Plant Physiol. 63: 341–345 Pegg AE (1988) Polyamine metabolism and its importance in neo-
Munro GF, Hercules K, Morgan J & Sauerbien W (1972) Depend- plastic growth as a target for chemotherapy. Cancer Res. 48:
ence of the putrescine content of E. coli on the osmotic strength 759–774
of the medium. J. Biol. Chem. 247: 1272–1280 Peleman J et al. (1989) Strong cellular preference in the expres-
Munro GF, Miller RA, Bell CA & Verdeber EL (1975) Effects sion of a housekeeping gene of Arabidopsis thaliana encoding
of external osmolarity on polyamine metabolism in HeLa cells. S-adenosyl-methionine synthetase. Plant Cell 1: 81–93
Biochim. Biophys. Acta 411: 263–281 Perez-Amador MA, Carbonell J & Granell A (1995) Expression
Murthy KS, Smith TA & Bould C (1971) The relation between the of Arginine decarboxylase is induced during early fruit devel-
putrescine content and potassium status of black currant. Ann. opment and in young tissues of Pisum sativum (L.). Plant Mol.
Bot. N.S. 35: 687–695 Biol. 28: 997–1009
Mussell H, Osmeloski JF, Wettlaufer SH & Weinstein LH (1987) Perez-Amador MA, Carbonell J, Navarro JL, Moritz T, Beale MH,
Suppression of Verticillium wilt of tomato by difluoromethyl- Lewis MJ & Hedden P (1996) N4 -Hexanoylspermidine, a new
orniltrine, a suicidal inhibitor of polyamine biosynthesis. Plant polyamine-related compound that accumulates during ovary and
Dis. 71: 313–316 petal senescence in pea. Plant Physiol. 110: 1177–1186
Nam KH, Lee SH & Lee JH (1996) A cDNA encoding arginine Perrella FW, Takigawa M & Boutwell RK (1983) Purification
decarboxylase (accession no. U35367) from soybean hypocotyls of 12-o-tetradecanoylphorbol-13-acetic induced orniltrine de-
(PGR 95-142). Plant Physiol. 110: 714 carboxylase from mouse epidermis. Int. J. Biochem. 15: 885–889
Nathan R, Altman A & Monselise SP (1984) Changes in activity Ploszaj T, Motyl T, Zimowska W, Skierski J & Zwierzchowski L
of polyamine biosynthetic enzymes and in polyamine contents (2000) Inhibition of ornithine decarboxylase by α-DFMO in-
in developing fruit tissues of ‘Murcott’ mandarin. Sci. Horticult. duces apoptosis in HC 11 mouse mammary epithelial cells.
22: 359–364 Amino Acids 19: 483–496
Noh E W & Minocha SC (1994) Expression of a human S- Poulin R, Lu L, Ackermann B, Bey P & Pegg AE (1992) Mechanism
adenosylmethionine decarboxylase cDNA in transgenic tobacco of the irreversible activation of mouse ornithine decarboxylase
and its effects on polyamine biosynthesis. Transgenic Res. 3: by α-difluoromethyl ornithine. J. Biol. Chem. 267: 150–158
26–35
32

Prakash L, John P, Nair GM & Prathapasenan G (1988) Effect of Rugini E (1992) Involvement of polyamines in auxin and Agrobac-
spermidine and methylglyoxal-bis(guanylhydrazone) (MGBG) terium rhizogenes induced rooting in fruit trees in vitro. J. Am.
on in vitro pollen germination and tube growth in Catharanthus Soc. Hort. Sci. 117: 532–536
roseus. Ann. Bot. 61: 373–375 Rugini E & Mencuccini M (1985) Increased yield in the olive with
Priebe A, Klein H & Jager HJ (1978) Role of polyamines in SO2 - putrescine treatment. Hortscience 20: 102–103
polluted pea plants. J. Exp. Bot. 29: 1045–1050 Samborski DJ & Rohringer R (1970) Abnormal metabol-
Pua EC (1993) Cellular and molecular aspects of ethylene on plant ites of wheat: occurrence, isolation and biogenesis of 2-
morphogenesis of recalcitrant Brassica species in vitro. Bull. hydroxyputrescine amides. Phytochemistry 9: 1939–1945
Bot. Acad. Sin. 34: 191–209 Santerra A, Markiewicz M & Villaneuva VR (1990) Effect of acid
Pua EC & Chi GL (1993) De novo shoot morphogenesis and rainon polyamines in Picea. Phytochemistry 29: 1767–1769
plant growth of mustard (Brassica juncea) in vitro in relation to Sawka MA, Niklas A & Jazdzewska (1997) The effect of polyam-
ethylene. Physiol. Plant 88: 467–474 ines on the development of sugar beet protoplasts. Biol. Plant 39:
Pua EC, Sim GE, Chi GL & Kong LF (1996) Synergistic effect 561–567
of ethylene inhibitors and putrescine on shoot regeneration from Sawka AM, Butowt R & Niklas A (1998) Do polyamines release
hypocotyl explants of Chinese radish (Raphanus sativus L. var. membrane-bound calcium in sugar beet protoplasts? J. Plant
longipinnatus Bailey) in vitro. Plant Cell Rep. 15: 685–690 Physiol. 153: 247–250
Purnhausser L, Medgysey M, Czeko PJ & Marton L (1987) Stimu- Schoofs G, Teichmann S, Hartmann T & Wink M (1983) Lysine de-
lation of shoot regeneration in Triticum aestivum and Nicotiana carboxylase in plants and its integration in quinolizidine alkaloid
plumbaginifolia Viv. Tissue culture using the ethylene inhibitor biosynthesis. Phytochemistry 22: 65–69
AgNO3 . Plant Cell Rep. 6: 1–4 Schroder G & Schroder J (1995) cDNAs for S-adenosyl-L-
Quattrocchio F, Benvenuto E, Tavazza L, Cuozzo L & Ancara G methionine decarboxylase from Catharanthus roseus, heterolog-
(1986) A study of the possible role of auxin in potato ‘hairy root’ ous expression, identification of the proenzyme-processing site,
tissues. J. Plant Physiol. 123: 143–150 evidence for the presence of both subunits in the active enzyme,
Rajam MV (1987) Polyamines. In: Prasad MNV (ed.) Plant Eco- and a conserved region in the 5 mRNA leader. Eur. J Biochem.
physiology (pp. 343–374). John Wiley and Sons, New York. 228: 74–78
Rajam MV (1993) Polyamine biosynthesis inhibitors: New protect- Schwartz M, Altman A, Cohen Y & Arzee T (1986) Localization
ants against fungal plant diseases. Curr. Sci. 65: 461–469 of ornithine decarboxylase and changes in polyamine content in
Rajam MV & Galston AW (1985) The effect of some polyamine root meristems of Zea mays. Physiol. Plant 67: 485–492
biosynthetic inhibitors on growth and morphology of phtopatho- Seeley JE, Poso H & Pegg AE (1982) Purification of ornithine
genic fungi. Plant Cell Physiol. 26: 683–692 decarboxylase from kidneys of androgen-treated mice. Biochem-
Rajam MV, Weinstein LH & Galston AW (1985) Prevention of a istry 21: 3394–3399
plant disease by specific inhibition of fungal polyamine biosyn- Seeley JE, Poso H & Pegg AE (1983) Labeling and quantita-
thesis. Proc. Natl. Acad. Sci. USA 82: 6874–6878 tion of ornithine decarboxylase protein by reaction with α-[5-
Rajam MV, Weinstein LH & Galston AW (1986) Kinetic studies on 14 C]difluoromethyl ornithne. In: Corbin JD & Hardman JG (eds)
the control of the bean rust fungus (Uromyces phaseoli L.) by an Methods in Enzymology. Polyamines, Vol. 94 (pp. 206–209).
inhibitor of polyamine biosynthesis. Plant Physiol. 82: 485–487 Academic Press
Rastogi R & Kaur-Sawhney V (1990) Polyamines and flower devel- Sharma P & Rajam MV (1995) Spatial and temporal changes in
opment in the male sterile stamen-2 mutant of tomato. 1. Levels the endogenous polyamine levels associated with embryogen-
of polyamines and their biosynthesis in normal and mutant esis from different hypocotyls segments of egg plant (Solanum
flowers. Plant Physiol. 93: 439–445 melongena). Plant Physiol. 146: 658–664
Rastogi R, Dulson J & Rothstein SJ (1993) Cloning of tomato (Ly- Shen W, Petit A, Guern J & Tempe J (1988) Hairy roots are more
copersicon esculentum Mill.) arginine decarboxylase gene and sensitive to auxin than normal roots. Proc. Natl. Acad. Sci. USA
its expression during fruit ripening. Plant Physiol. 103: 829–834 85: 3417–3421
Recsei PA & Snell EE (1984) Pyruvoyl enzymes. Ann. Rev. Bio- Shevyakova NI (1966) Influence produced by salts on biosynthesis
chem. 53: 357–387 of certain sulphur-containing amino acids in leaves of vicia faba.
Richards FJ & Coleman RG (1952) Occurrence of putrescine in Fiziol. Rast. 13: 522–524
potassium deficient barley. Nature 170: 460 Shiozaki S, Ogata T & Horiuchi T (2000) Endogenous polyamines
Rinaldi A, Floris G & Finazzi-Agro A (1982) Purification and prop- in the pericarp and seed of the grape berry during ripening. Sci.
erties of diamine oxidase from Euphorbia latex. Eur. J. Biochem. Hort. 83: 33–41
127: 417–422 Sindhu RK & Cohen SS (1984) Subcellular localisation of sper-
Roberts DR, Walker MA, Thompson JE & Dumbroff EB (1984) midine synthase in the protoplasts of chinese cabbage leaves.
The effect of inhibitors of polyamine and ethylene biosynthesis Plant Physiol. 76: 219–223
on senescence, ethylene production and polyamine levels in cut Sindhu RK & Desai HV (1979) Purification and properties of
carnations. Plant Cell Physiol. 25: 315–322 agmatine aminohydrolase from groundnut cotyledons. Phyto-
Roberts DR, Dumbroff EF & Thompson JE (1986) Exogenous chemistry 18: 1937–1938
polyamines alter membrane fluidity in bean leaves-a basis for po- Slocum RD (1991) Polyamine biosynthesis in plants. In: Slo-
tential misinterpretation of their true physiological role. Planta. cum RD & Flores HE (eds) Biochemistry and Physiology of
167: 395–401 Polyamines in Plants (pp. 23–40). CRC Press, Boca Raton, FL,
Robie C & Minocha SC (1989) Polyamines and somatic embryo- Slocum RD & Galston AW (1985a) Changes in polyamine biosyn-
genesis in carrot. I. The effects of difluoromethylornithine and thesis associated with postfertilization growth and development
difluoromethylarginine. Plant Sci. 65: 45–54 in tobacco ovary tissues. Plant Physiol. 79: 336–343
Robins DJ & Walters DR (1995) Antifungal compounds. US Patent Slocum RD & Galston AW (1985b) In vivo inhibition of polyam-
5461079 ine biosynthesis and growth in tobacco ovary tissues. Plant Cell
Physiol. 26: 1519-1526
 33

Slocum RD, Kaur-Sawhney R & Galston AW (1984) The Stevans L & Stevans E (1980) Role of polyamines in higher
physiology and biochemistry of polyamines in plants. Arch. plants and their biomedical applications. In: Gaugus JM (ed)
Biochem. Biophys. 235: 283–303 Polyamines in Biomedical Research (pp. 167–183). Wiley, New
Slocum RD, Bionti AJ, McCann PP & Feirer PP (1988) DL- York
α-Difluoromethyl [3,4-3 H]arginine metabolism in tobacco and Stroinski A & Szezotka Z (1989) Effect of cadmium and Phytoph-
mammalian cells. Biochem. J. 255: 197–202 thora infestans on polyamine levels in potato leaves. Physiol.
Slotkin TA, Ferguson SA, Cada AM, McCook EC & Seidler FJ Plant 77: 244–246
(2000) Neonatal polyamine depletion by α-DFMO: effects on Sun Li-Y, Monneuse M-O, Josette MT & Tepfer D (1991) Changes
adenylyl cyclase cell signalling are separable from effects on in flowering and accumulation of polyamines and hydrocinnamic
brain region growth. Brain Res. 887: 16–22 acid-polyamine conjugates in tobacco plants transformed by the
Smith TA (1963) L-Arginine carboxylase of higher plants and its rolA locus from the Ri TL-DNA of Agrobacterium rhizogenes.
relation to potassium nutrition. Phytochemistry 2: 241–252 Plant Sci. 80: 145–156
Smith MA & Davies PJ (1985) Effect of photoperiod on polyamine Suresh MR & Adiga PR (1977) Putescine-sensitive (artifac-
metabolism in apical buds of g-2 peas in relation to the induction tual) and insensitive (biosynthetic) S-adenosyl-L-methionine de-
of apical senescence. Plant Physiol. 79: 400–405 carboxylase activities of Lathyrus sativus seedlings. Eur. J Bio-
Smith TA (1965) N-Carbamylputrescine amidohydrolase of higher chem. 79: 511–518
plants and its relation to potassium nutrition. Phytochemistry 4: Suresh MR, Ramakrishna S & Adiga PR (1978) Regulation of ar-
599–607 ginine decarboxylase and putrescine levels in Cucumis sativus
Smith TA (1970) Polyamine oxidase in higher plants. Biochim. cotyledons. Phytochemistry 17: 57–63
Biophys. Res. Commun. 41: 1452–1456 Suttle JC (1981) Effect of polyamines on ethylene production.
Smith TA (1979) Arginine decarboxylase of oat seedlings. Phyto- Phytochemistry 20: 1477–1480
chemistry 18: 1447–1452 Tabor CW & Tabor H (1984) Polyamines. Annu. Rev. Biochem. 53:
Smith TA (1981) Amines. In: Stumpf PK & Conn EE (eds) The 749–790
Biochemistry of Plants, Vol. 7 (pp. 249–268). Academic Press, Tabor CW & Tabor H (1985) Polyamines in microorganisms.
New York Microb. Rev. 49: 81–89
Smith TA (1983) Polyamine oxidase (oar seedlings). In: Tabor H & Tassoni A, Antognoni F & Bagni N (1996) Polyamine binding to
Tabor CW (eds) Methods in Enzymology, Vol. 94 (pp. 311–314). plasma membrane vesicles isolated from Zucchini hypocotyls.
Academic Press, New York Plant Physiol. 110: 817–824
Smith TA (1984) Putrescine and inorganic ions. Adv. Phytochem. Tassoni A, Antognoni F, Bastistini ML, Sanvido O & Bagni N
18: 7–54 (1998) Characterization of spermidine binding to solubilized
Smith TA (1985) Polyamines. Annu. Rev. Plant Physiol. 36: 1611– membrane proteins from Zucchini hypocotyls. Plant Physiol.
1613 117: 971–977
Smith TA (1993) Amines. In: Harborne JB & Dey PM (eds) Meth- Taylor MA et al. (1992) Expression and sequence analysis of
ods in Plant Biochemistry 8 (Alkaloids and Sulphur compounds) cDNAs induced during the early stages of tuberisation in differ-
(pp. 17–49). Academic Press ent organs of potato plant (Solanum tuberosum). Plant Mol. Biol.
Smith TA & Best GR (1978) Distribution of the hordatines in barley. 20: 641–651
Phytochemistry 17: 1093–1098 Teitel DC, Cohen E, Arad S (M), Birnbaum E & Mizrahi Y (1985)
Smith TA & Sinclair C (1967) The effect of acid feeding on amine The possible involvement of polyamines in the development of
formation in barley. Ann. Bot. (N.S.) 31: 103–111 tomato fruits in vitro. Plant Growth Regul. 3: 309–317
Smith TA, Negrel J & Bird CR (1983) The cinnamic acid amides Tepfer D (1982) La transformation genetique de plantes su-
of the di- and polyamines. In: Bachrach U, Kaye A & Chayen perieures par Agrobacterium rhizogenes. In: 2E Colloque sur les
R (eds) Advances in Polyamine Research, Vol. 4 (pp. 347–370). Recherches Fruitieres. Centre Technique Interprofessionnel des
Raven Press, New York Fruits et Legumes, Bordeaux, France (pp. 47–59)
Songstad DD, Armstrong CL & Peterson WL (1991) AgNO3 in- Tepfer D (1984) Transformation of several species of higher plants
creases type II callus production from immature embryos of by Agrobacterium rhizogenes: sexual transmission of the trans-
maize inbred B73 and its derivatives. Plant Cell Rep. 9: 699–702 formed genotype and phenotype. Cell 47: 959–967
Soyka S & Heyer AG (1999) Arabidopsis knockout mutation of Terano S & Suzuki Y (1978) Formation of β-alanine from spermine
ADC2 gene reveals inducibility by osmotic stress. FEBS Lett. and spermidine in maize shoots. Phytochemistry 17: 148–149
458: 219–223 Tiburcio AF, Kaur-Sawhney R & Galston AW (1987) Effect of
Spano L, Mariotti D, Cardarelli M, Branca C & Costantino P (1988) polyamine biosynthetic inhibitors on alkaloids and organogen-
Morphogenesis and auxin sensitivity of transgenic tobacco with esis in tobacco callus cultures. Plant Cell Tissue Org. Cult. 9:
different complements of Ri T-DNA. Plant Physiol. 87: 479–483 111–120
Spernaza A, Calzoni GL & Bagni N (1983) Effect of exogenous Tiburcio AF, Gendy CA & Tran Than Van K (1989) Morphogenesis
polyamines on in vitro germination of apple pollen. In: Mulcahy in tobacco subepidermal cells: Putrescine as a marker of root
DL & Ottavio (eds) Pollen: Biology and Implications for Plant differentiation. Plant Cell Tissue Org. Cult. 19: 43–54
Breeding (pp. 21–103). Elsevier Biomedical, New York. Tiburcio AF, Kaur-Sawhney R & Galston AW (1990) In: Miflin
Srivastava SK, Vashi DJ & Naik BI (1983) Control of senescence by BJ & Lea PJ (eds) Intermediary Nitrogen Metabolism (pp.
polyamines and guanidines in young and mature barley leaves. 283–325). Academic Press, New York
Phytochemistry 22: 2151–2154 Tiburcio AF, Figueras X, Claparols I, Santos M & Torne JM (1991)
Srivenugopal KS & Adiga PR (1981) Enzymic conversion of ag- Improved plant regeneration in maize callus cultures after pre-
matine to putrescine in Lathyrus seedlings. Purification and treatment with DL-α-difluoromethyl arginine. Plant Cell Tissue
properties of a multifunctional enzyme (putrescine synthase). J Org. Cult. 27: 27–32
Biol. Chem. 256: 9532–9541 Torget R, Lapi L & Cohen SS (1979) Synthesis and accumulation
of polyamines and S-adenosyl methionine in chinese cabbage
34

infected by turnip yellow mosaic virus. Biochem. Biophys. Res. Wang SY & Steffens GL (1985) Effect of paclobutrazol on water
Commun. 87: 1132–1139 stress induced ethylene biosynthesis and polyamine accumula-
Torne JM, Claparols I, Marce M, Guergue AM & Santos MA (1994) tion in apple seedling leaves. Phytochemistry. 24: 2185–2190
Influence of pretreatments with inhibitors of putrescine synthesis Wang SY, Faust M & Steffens GL (1985) Metabolic changes in
on polyamine metabolism and differentiation processes of maize cherry flower buds associated with breaking of dormancy in early
calluses. Plant Sci. 100: 15–22 and late blooming cultivars. Physiol. Plant 65: 89–94
Torrigiani P, Serafini-Fraccasini D & Bagni N (1987a) Polyamine Watson MB & Malmberg RL (1996) Regulation of Arabidop-
biosynthesis and the effect of dicyclohexylamine during the cell sis thaliana (L.) Heysh arginine decarboxylase by potassium
cycle of Helianthus tuber. Plant Physiol. 84: 148–152 deficiency stress. Plant Physiol. 111: 1077–1083
Torrigiani P, Altamura MM, Pasqua G, Monacelli B, Serafini- Weinstein LH & Galston AW (1989) Prevention of a plant disease by
Fracassini D & Bagni N (1987b) Free and conjugated polyam- specific inhibition of fungal polyamine biosynthesis. US Patent
ines during de novo floral and vegetative bud formation in thin 4818770
cell-layers of tobacco. Physiol. Plant 70: 453–460 Weinstein LH, Osmeloski JF, Wettlaufer SH & Galston AW (1987)
Tran Thanh Van KM (1973) Direct flower formation from superfi- Protection of wheat against leaf and stem rust and powdery mil-
cial tissues of small explant of Nicotiana tabacum. Planta 115: dew diseases by inhibition of polyamine metabolism. Plant Sci.
87–92 51: 311–316
Tran Thanh Van KM (1981) Control of morphogenesis in in vitro West HM & Walters DR (1989) Effects of polyamine biosynthesis
cultures. Annu. Rev. Plant Physiol. 32: 291–311 inhibitors on growth of Pyrenophora teres, Gaeumannomyces
Tyagi AK, Tabor CW & Tabor H (1981) Ornithine decarboxylase graminis, Fusarium culmorum and Septoria nodorum in vitro
from Saccharomyces cerevisiae: Purification, properties and Mycol. Res. 92: 453–457
regulation of activity. J. Biol. Chem. 256: 12156–12163 Winer L & Apelbaum A (1986) Involvement of polyamines in the
Vain P, Flament P & Soudain P (1989) Role of ethylene in em- development and ripening of avocado fruits. J. Plant Physiol. 26:
bryogenic callus initiation and regeneration in Zea mays. J. Plant 223–234
Physiol. 135: 537–540 Wu SC & Kuniyuki AH (1985) Isolation and culture of almond
Van den Broeck D, Van Den Straeten D, Van Montague M & protoplasts. Plant Sci. 41: 55–60
Caplan A (1994) A group of chromosomal proteins is specific- Yadav JS & Rajam MV (1997) Spatial distribution of free and
ally released by spermine and loses DNA-binding activity upon conjugated polyamines in leaves of Solanum melongena asso-
phosphorylation. Plant Physiol. 106: 559–566 ciated with differential morphogenetic capacity efficient somatic
Velikova V, Yordanov I & Edreva A (2000) Oxidative stress and embryogenesis with putrescine. J. Exp. Bot. 48: 1537–1545
some antioxidant system in acid rain-treated bean plants. Pro- Yanasigawa H & Suzuki Y (1982) Purification and properties
tective role of exogenous polyamines. Plant Sci. 151: 59–66 of N-carbamoyl-putrescine aminohydrolase from maize shoots.
Volz RK & Knight JN (1986) The use of growth regulators to Phytochemistry 21: 2201–2203
increase precocity in apple trees. J. Hort. Sci. 61: 181–189 Yanasigawa H, Hirasawa E & Suzuki Y (1981) Purification and
Walker MA, Roberts DR, Shih CY & Dumbroff EB (1985) A properties of diamine oxidase from pea epicotyls. Phytochem-
requirement for polyamines during the cell division phase of rad- istry 20: 2105–2108
icle emergence in seeds of Acer saccharum. Plant Cell Physiol. Yang SF & Hoffman NE (1984) Ethylene biosynthesis and its regu-
26: 967–972 lation in higher plants. Annu. Rev. Plant Physiol. 35: 155–189
Walters DR (1987) Polyamines: the cinderellas of cell biology. Ye B, Muller HH, Zhang J & Gressel J (1997) Constitutively el-
Biologist 34: 73–76 evated levels of putrescine and putrescine-generating enzymes
Walters DR (1989) Polyamines and plant disease. Plants Today correlated with oxidant stress resistance in Conzya bonariensis
(Jan–Feb), 22–26 and wheat. Plant Physiol. 115: 1443–1451
Walters DR & Wylie MA (1986) Polyamines in discrete regions of Young ND & Galston AW (1983a) Are polyamines transported in
barley leaves infected with the powdery mildew fungus, Erysiphe etiolated peas? Plant Physiol. 73: 912–914
graminis. Physiol. Plant 67: 630–633 Young ND & Galston AW (1983b) Putrescine and acid stress. Plant
Wang SY (1987) Changes of polyamines and ethylene in cucumber Physiol. 71: 767–771
seedlings in response to chilling stress. Physiol. Plant 69: 253– Young ND & Galston AW (1984) Physiological control of arginine
257 decarboxylase activity in potassium deficient oat shoots. Plant
Wang SY & Faust M (1986) Effect of growth retardants on root Physiol. 76: 331–335
formation and polyamine content in apple seedlings. J. Am. Hort.
Sci. 111: 912–917