J. Plant Physiol. Vol. 142. pp.

81-87 (1993)

Regulation of Rhizogenesis by Polyamines in Tobacco Thin

Layers

PATRIZIA TORRIGIANI
1
*, 2
MARIA MADDALENA ALTAMURA , SONIA SCARAMAGLI\
2 2 1
FRANCESCA CAPITANI , GIUSEPPINA FALASCA , and NELLO BAGNI
1 Dipartimento di Biologia e.s., Universid di Bologna, Via Irnerio 42, 40126 Bologna (Italy)
2 Dipartimento di Biologia Vegetale, Universitii La Sapienza, P.le Aldo Moro, 00185 Roma (Italy)
* To whom correspondence should be addressed.

Received November 10, 1992 . Accepted February 25,1993

Summary

Tobacco thin layer explants were cultured on a rhizogenic medium for 21 days in the presence or ab-
sence of methylglyoxal-bis(guanylhydrazone) (MGBG) or cyc10hexylamine (CHA), competitive inhib-
itors of spermidine synthesis through S-adenosylmethionine decarboxylase and spermidine synthase, re-
spectively. On day 21, explants were transferred to hormone-free medium of the same composition
with or without MGBG or CHA, alone or in combination with labelled and unlabelled spermidine.
The effects of these treatments on free and bound polyamine levels, on the putrescine biosynthetic ac-
tivity (ornithine decarboxylase, ODC, and arginine decarboxylase, ADC), on labelled spermidine in-
corporation and on the rhizogenic response were studied. In MGBG-treated explants rhizogenesis was
strongly inhibited while with CHA it was less affected. In the former, the addition of spermidine caused
a significant reversion of the rooting inhibition (considering both rooting percentage and the mean num-
ber of roots per explant), while not in the latter. MGBG induced strong putrescine and spermidine deple-
tion both in the free and bound forms; CHA induced strong spermidine depletion (especially on day 21),
but putrescine accumulation. In both treatments, in the presence of labelled spermidine, labelled free and
bound putrescine were also detected.
A general enhancement in ADC activity was observed in inhibitor-treated explants; the addition of
spermidine to the inhibitors caused a further strong increase in ADC activity. These results are discussed
in relation to the different metabolic pathways affected by the inhibitors.

Key words: A rginine decarboxylase, Nicotiana tabacum, ornithine decarboxylase, putrescine, spermidine,
spermidine biosynthesis inhibitors, thin layers, rhizogenesis.

Abbreviations: ADC = arginine decarboxylase; CHA = cyc1ohexylamine; MGBG = methylglyoxal-

bis(guanylhydrazone); ODC = ornithine decarboxylase; SAMDC = S-adenosylmethionine decarboxyl-
ase; TCA = trichloroacetic acid.

Introduction polyamines (Bagni et al., 1993; Bagni and Torrigiani, 1992).

Evidence increasingly supports the view that polyamines,
beyond their role in cell division, are also involved in organ-
ogenIC processes.
In fact, changes in the state of differentiation of cells are al-
ways associated with changes in the levels of free and bound
This is also confirmed by the fact that inhibition of poly-
amine biosynthesis, at various levels of the metabolic path-
way, affects morphogenic processes in all the plant systems
studied (Galston and Flores, 1991; Bagni et al., 1993).
We focused our attention on the rhizogenic process in to-
bacco thin layers. In this model system the levels of free and

© 1993 by Gustav Fischer Verlag, Stuttgart

82 P. TORJlIGIANI, M. M. ALTAMURA, S. SCARAMAGU, F. CAPITANI, G. FALASCA, and N. BAGNI
bound polyamines increase early in culture and continue to
rise during the period of root meristemoid organization,
which leads to root formation (T orrigiani et aI., 1989). The
inhibition of polyamine biosynthesis by specific or competi-
tive inhibitors depresses polyamine titers and rhizogenesis to
different extents depending on the type of drug (Altamura et
aI., 1991). In particular, as far as treatment with the inhib-
itors of spermidine biosynthesis, methylglyoxal-bis(guanyl-
hydrazone) (MGBG, inhibitor of S-adenosylmethionine de-
carboxylase activity) and cyclohexylamine (CHA, inhibitor
of spermidine synthase activity), is concerned, in the former,
rhizogenesis is strongly inhibited, while spermidine and put-
rescine titers first decline and then increase dramatically; in
the latter, rhizogenesis is not strongly affected while spermi-
dine is depleted and putrescine accumulates (Altamura et aI.,
1991). In both cases non-dividing hypertrophic cells are ob-
served in the explants within 15-18 days in culture (Bagni et
al., 1993), concomitantly with maximum spermidine deple-
tion (Altamura et aI., 1991); spermidine, supplied with the
drug at the beginning of culture, prevents the occurrence of
these events (Bagni et al., 1993).
In the present work, rhizogenic tobacco thin layer ex-
plants were transferred on day 21 to hormone-free medium
containing spermidine as well as CHA or MGBG in order to
avoid interactions between the hormones and the polyamine
during the last phases of the rhizogenic process. The aim was
to establish a possible spermidine-induced reversion of inhi-
bition of rhizogenesis, to better clarify the putrescine bio-
synthesis and accumulation induced by the inhibitors of
spermidine synthesis and to verify a possible inverse spermi-
dine-putrescine metabolic link. For this purpose, on day 21,
which corresponded to the time of its maximum depletion
(Torrigiani et aI., 1989), labelled and unlabelled exogenous
spermidine was added with CHA or MGBG at a concentra-
tion comparable to the physiological one measured in con-
trols on that day. The effect of these treatments on free and
bound polyamine levels, on the putrescine biosynthetic ac-
tivity (ornithine decarboxylase, ODC, and arginine decar-
boxylase, ADC), on labelled spermidine incorporation and
on the rhizogenic response was studied.
Materials and Methods
Plant material and tissue culture
Plants of Nicotiana tabacum L. cv. Samsun were grown in the
field up to the complete green fruit stage. Thin layer explants
(2 x 10 mm, mean fresh weight 11.7 ± 1.3 mg) consisting of 8 cell
layers (epidermal, subepidermal and parenchyma cells) were excised
from the stem (between the 5th and 8th node from the lowest axil-
lary inflorescence), sterilized and cultured on a root-inducing me-
dium, as previously described (Torrigiani et a!., 1989).
The medium also contained either 2.5 mM CHA or 0.5 mM
MGBG. On day 21 all the explants were transferred to a hormone-
free medium of the same composition or containing, in addition,
2.5 mM CHA and 0.3 mM MGBG, respectively, alone or in combi-
nation with OAmM spermidine and 370Bq/mL [1,4-
14C]spermidine (specific activity 4.07 GBql nmol). The cultures
were grown for 30 days at 27 ± 1 °C in continuous darkness. The
time course of morphogenesis was followed on stocks of 200 (up to
day 21 for inhibitor treatments) or 75 (controls, inhibitor- and in-
hibitor plus spermidine treatments from day 21 to 30) explants per
treatment by observation under a stereo microscope starting from
day 10. On days 0, 21 and 30 explants were harvested, pooled,
weighed and stored at - 80°C until use.
Data on the percentage of rooting explants were analyzed statisti-
cally using the X- test and those on the mean number of roots per
explant by Student's t test.
Polyamine extraction and analysis
Samples were analyzed for free, TCA-soluble and TCA-insoluble
bound polyamines. They were homogenized in 5 % TCA and cen-
trifuged for 15 min at 26,000 x g, to allow the recovery of a superna-
tant (SN) and pellet (PT). The pellets were resuspended in the origi-
nal volume of TCA. Replicates (0.2 mL) of this suspension and of
the supernatant were placed in glass ampoules with the same vol-
ume of 12N HCl; the ampoules were then flame sealed and in-
cubated at 110°C for 18 h to allow the hydrolysis of covalent link-
ages between polyamines and other molecules. The hydrolysates
were taken to dryness and resuspended in 0.2 mL TCA. Aliquots
(0.1 mL) of the TCA supernatant (SN), hydrolyzed supernatant
(SNi) and hydrolyzed resuspended pellet (PTi) were dansylated and
dansyl-polyamines extracted in benzene as previously described
(Torrigiani et a!., 1989). Spots, visualized under UV light, were
scraped off, eluted in acetone and either measured for fluorescence
using a Jasco FP-770 spectrofluorimeter (excitation 360 nm, emis-
sion 506 nm) and compared with that of dansylated standards, or
measured for radioactivity in Ready Gel scintillation cocktail (Beck-
man) using a LS-1800 (Beckman) scintillation counter. Total radio-
activity was also measured during the various phases of polyamine
extraction and separation.
ADC and ODC activity assay
All extraction procedures were carried out on ice. Explants
(100-5OOmg) were homogenized in five volumes of Tris-HCl
100 mM, pH 8.5, containing 50 mM pyridoxal phosphate (PLP), ac-
cording to the optimized procedure described by Altamura et al.
(1993). ADC (EC 4.1.1.19) and ODC (EC 4.1.1.17) activity assays
consisted in measuring the HC0 2 evolution from 7.4 kBq of L-[l-
14C]ornithine (1.93GBqmmol-1, Amersham) or of DL-[l-
14C]arginine (12A3GBqmmol-1, CEA), as previously described by
Torrigiani et a!. (1987).
Protein content was measured using the protein-dye binding
method of Bradford (1976) with bovine serum albumin as the stand-
ard.
Results
Time course 0/ rhizogenesis
The first roots, regular in size and shape, occasionally ap-
peared in control explants on day 13 (Table 1), in inhibitor-
treated ones on day 15. On the same day controls showed 6-
and 4-fold more rooting explants than MGBG- and CHA-
treated ones, respectively. On day 18 the percentage of root-
ing explants increased in all treatments, although in MGBG-
and CHA-treated cultures it remained lower than in control
explants.
On day 26, 5 days after transfer to hormone-free medium,
the number of rooting explants continued to increase with
respect to day 18 in controls, but not in MGBG or CHA
treatments.
 Rhizogenesis regulation by polyamines 83

Table 1: Time course of morphogenesis expressed as percentage of tobacco thin layer explants cultured with or without polyamine bio-
synthesis inhibitors. CR, callus plus roots; C, only callus; S, swollen; U, unchanged. MGBG, methylglyoxal-bis(guanylhydrazone); CHA,
cyclohexylamine; Sd* indicates the presence of labelled (370 Bq/ mL) plus unlabelled (0.4 mM) spermidine in the medium.
Control MGBG CHA
Days U S C CR U S C CR U S C CR
10 26.7 33.3 40.0 0 62.0 29.5 8.5 0 47.5 30.5 22.0 0
13 26.7 16.0 56.0 1.3 48.5 28.0 23.5 0 21.5 41.5 37.0 0
15 14.7 18.7 26.7 39.9 40.5 29.5 23.5 6.5 20.0 29.0 41.0 10.0
18 14.7 10.7 30.7 43.9 28.0 33.5 28.5 10.0 19.5 26.0 34.5 20.0
21 Transplantation in hormone-free medium
+Sd* 28.0 13.3 32.0 26.7 13.3 18.6 41.4 26.7
26
none 8.0 4.0 12.0 76.0 29.4 42.7 17.3 10.6 13.3 21.3 36.0 29.4
+Sd" 26.7 13.3 12.0 48.0 13.3 9.3 17.3 60.1
30
none 4.0 0 5.4 90.6 26.7 41.3 10.7 21.3 10.6 24.0 10.7 54.7

Already at this time, spermidine caused a significant (P
< 0.05) reversion of the inhibition of rhizogenesis (recovery
of rooting ability 2.5-fold) in MGBG-treated explants, but
not in CHA-treated ones (Table 1).
By day 30, a significantly higher (P < 0.Q1) percentage of
MGBG+spermidine-treated explants produced roots than
those treated with MGBG alone; this percentage, however,
was significantly lower (P < 0.01) than that of controls. No
significant reversion of the CHA-induced inhibition by sper-
midine was observed.
The number of root-forming explants was higher in the
presence of CHA than of MGBG (P < 0.Q1), while no signif-
icant difference was observed when the drugs were supplied
with spermidine.
The pattern of callogenesis (Table 1) correlated with that
of rhizogenesis in that the percentage of explants with only
callus generally decreased with the progression of rhizogene-
sis. At the end of the culture period the highest number of
unchanged explants was observed in MGBG treatments in-
dependently of the final percentage of rooting explants. In
the treatment with MGBG alone the highest percentage of
swollen explants was also observed. The addition of spermi-
dine induced a highly significant reduction (P < 0.Q1) in the
number of swollen explants observed on day 30 with respect
to the treatments with the inhibitors alone (Table 1).
The results on rooting percentage are supported by data
on the number of roots per explant at the end of the culture
period. Control explants produced a significantly higher
number of roots per explant (10.3 ± 1.02) than MGBG- (3.5
± 0.48, P < 0.Q1) and CHA-treated ones (7.32 ± 0.61, P
<0.05). MGBG+spermidine gave a significantly higher
number of roots per explant (6.43 ± 0.83, P <0.05) than
MGBG alone, while CHA+spermidine (8.98 ± 1.00) did
not significantly increase that value with respect to the in-
hibitor alone.
Changes in the fresh weight of explants were strongly de-
pendent on the amount of callus. On day 21 the fresh weight
of MGBG- and CHA-treated explants (129.6 ± 15.4 and
125.7 ± 14.0 mg, respectively) was comparable to that of
controls (116.5 ± 5.4 mg). On day 30, control explants, most
of which had roots, exhibited a fresh weight of 166.1 ±
41.3 mg, while the MGBG-treated (278.5 ± 48.0 mg) and es-
pecially the CHA-treated explants (513.6 ± 84.7 mg) were
more conspicuous, due to the presence of callus. Spermidine,
added to the inhibitors, caused a reduction in callus forma-
tion and an enhancement in rooting response; thus the fresh
weight of explants (MGBG+spermidine: 151.9 ± 9.4 mg;
CHA+spermidine: 167.5 ± 50.9 mg) was lower than that of
the treatments with the inhibitors alone and comparable to
that of controls.
Free and bound polyamine titer
In control explants increased levels of TCA-soluble free,
TCA-soluble bound and TCA-insoluble bound putrescine
and spermidine were detected on day 21 in culture, i.e. at the
end of the culture phase in the presence of hormones, while
spermine was not detectable (Table 2). The increase was par-
ticularly dramatic for the conjugated forms (139-fold TCA-
soluble bound putrescine, 19-fold TCA-soluble bound sper-
midine; 47-fold TCA-insoluble bound putrescine, 15-fold
TCA-insoluble bound spermidine).
MGBG caused an inhibition of free and TCA-insoluble
bound polyamine accumulation while the level of TCA-sol-
uble bound polyamines was not significantly affected (Table
2, day 21).
On the same day CHA caused spermidine depletion par-
ticularly in the TCA-soluble and -insoluble bound forms.
Putrescine content was also depleted with respect to con-
trols, especially in the free and TCA-insoluble bound forms.
This trend generally resembles that previously described for
the same root-forming explants at the same time in culture
(Altamura et ai., 1991), although the absolute values are
slightly different.
Results concerning polyamine titers on day 30 must be
considered in the light of the fact that from days 21 to 30, ex-
plants were cultured on hormone-free medium. In controls,
putrescine, in all classes as well as bound spermidine, was
lower than on day 21 (Table 2). In MGBG-treated explants
putrescine and spermidine levels were even more strongly
depleted; in particular, they were no longer detectable in the
bound forms. CHA induced a further depletion in free but
84 P. TORRIGIANI, M. M. ALTAMURA, S. SCARAMAGLI, F. CAPITANI, G. FALASCA, and N. BAGNI

Table 2: TCA-soluble free (TSF), TCA-soluble bound (TSB) and Table 3: Distribution of radioactivity, expressed as total dpm in
TCA-insoluble bound (TIB) polyamines [nmol(gFW)-l] during a 500 mg-sample, on day 30 during polyamine extraction and deter-
rhizogenesis in tobacco thin layer explants cultured with or without mination in tobacco thin layer explants cultured from days 21 to 30
inhibitors. On day 21 explants were transferred to hormone-free on hormone-free medium containing MGBG+Sd* or CHA+Sd*.
medium. Sd* indicates the presence of labelled plus unlabelled sper- Figures are the means of two replicates. Sd* indicates the presence of
midine added to the medium on day 21. Pu, putrescine; Sd, spermi- labelled plus unlabelled spermidine in the medium. SN, superna-
dine; n.d. = not detectable. Figures represent the means ± SE of tant; PT, pellet; SNi, hydrolyzed supernatant; PTi, hydrolyzed re-
4-6 replicates (two experiments). suspended pellet; Pu putrescine; Sd, spermidine.
TSF TCA Benzene phase Pu+Sd
Days 0 21 30 Treatment SN PT SN SNi PTi SN SNi PTi
Pu Sd Pu Sd Pu Sd MGBG+Sd* 42480 4480 20800 25600 700 6250 6350 n.d.
Control 14±3 25±2 90± 17 115± 11 37± 2 161± 9 CHA+Sd* 37776 5152 23400 27400 1820 11400 8820 n.d.
MGBG 14±3 25±2 33± 4 50±13 9± 2 27± 1
CHA 14±3 25±2 50± 9 50±12 70± 6 14± 2
MGBG+Sd* 79±18 1006±94
CHA+Sd* 109± 10 1182±82 Table 4: Labelled polyamine content, expressed as Bq (gFW)-l, in
TSB tobacco thin layers cultured from days 21 to 30 on hormone-free
medium containing MGBG+Sd or CHA+Sd. Sd* indicates the
Control 6±1 11±2 835±209 212±70 260±27 n.d.
presence of labelled and unlabelled spermidine in the medium. Pu,
MGBG 6±1 11±2 1001 ± 102 252±21 n.d. n.d.
putrescine; Sd, spermidine; TSF, TCA-soluble free; TSB, TCA-solu-
CHA 6±1 11±2 624± 10 10± 8 593±76 112± 4
n.d. ble bound; n.d. = not detectable. Figures represent the means ± SE
MGBG+ Sd* 499±71
CHA+Sd* 489±81 94±10 of 4-6 replicates (two experiments).
Till TSF TSB
Control 18±5 16±2 839± 124 235±40 114± 3 35± 2 Treatment Sd Pu Sd Pu
MGBG 18±5 16±2 151± 28 36± 1 n.d. n.d.
MGBG+Sd* 361±58 33±6 n.d. 182±11
CHA 18±5 16±2 280± 30 10± 3 529±12 43± 5
CHA+Sd* 437±30 n.d. 216±6 111 ±22
MGBG+Sd* 114± 3 30± 8
CHA+Sd* 138± 16 24± 6

phase as dansylated polyamines and lipophilic compounds;

an increase in conjugated spermidine content. Putrescine
content was higher in all cases than in controls.
In the MGBG+spermidine treatment, the exogenously
supplied spermidine (0.4 mM) was taken up and concen-
trated in the free form (about 1 mM), but not in the conju-
gated ones; on the contrary, free putrescine content did not
increase much while TCA-soluble and -insoluble bound pu-
trescine increased noticeably with respect to the MGBG-
treated cultures. The CHA + spermidine treatment also led
to free spermidine accumulation, while putrescine did not
accumulate significantly with respect to the CHA treatment.
The dansylderivative of CHA was also measured by TLC
as previously described (Torrigiani et al., 1987). In CHA-
treated explants the drug accumulated (7.33 ~mol per g FW
on day 21) and then decreased on day 30 (3.1 ~mol per g
FW); in the CHA+spermidine treatment (which started on
day 21) the CHA accumulated by day 30 was the same
(6.52 ~mol per g FW) as in the CHA treatment on day 21.
Labelling experiments
14
The label distribution in [ C]-spermidine-fed explants was
checked during polyamine extraction and analysis. No sig-
nificant differences were found in the two inhibitor treat-
ments in the accumulation and partitioning of label between
the TCA supernatant and pellet (Table 3); 9.5 % (MGBG+
spermidine), and 12% (CHA+spermidine) of the total in-
corporated radioactivity was recovered in TCA-precipitable
molecules. In MGBG + spermidine cultures 49 % of the label
present in the supernatant was extracted in the benzene
30 % of the label present in the benzene phase of the super-
natant was due to labelled free polyamines. A slightly higher
amount of label (60 %) was extracted in benzene after hydro-
lysis and 25 % of this was represented by labelled polyamines
(Table3).
With respect to MGBG+spermidine, more label was ex-
tracted in the benzene phase of the supernatant (62 %), hy-
drolyzed supernatant (74%) and hydrolyzed pellet (35%) of
CHA + spermidine-treated explants. Of the label in the su-
pernatant extracted in benzene, 49 % and 37 % were due to
free polyamines. The amount of labelled polyamines recov-
ered after hydrolysis was not significantly different from
that of free polyamines; consequently, no TCA-soluble and
-insoluble bound polyamines were detectable.
The presence of labelled bound polyamines, although not
detectable as the difference between total label recovered in
the benzene phase of hydrolyzed and non-hydrolyzed super-
natants, was, however, appreciable in the single spots on
TLC plates (Table 4). As expected in MGBG+spermidine
and CHA + spermidine treatments, free and/or TCA-soluble
e
bound 4C]spermidine was recovered. In both cases, more-
over, [14C]putrescine was also present in free and/or bound
form.
ADC and ODC activities
On day 21, both ADC and ODC activities were signifi-
cantly enhanced by CHA and only slightly by MGBG with
respect to controls (Table 5). At the end of the culture pe-
riod (day 30) ADC activity was enhanced by both MGBG

Table 5: ADC and ODC activity expressed as pmol(mgprot.t l h- 1
in tobacco thin layer explants cultured with or without inhibitors.
Values are the means ± SE of 3-4 replicates. On day 0, ADC activ-
ity was 191 and ODC 366 pmol (mgprot.t I h- 1• On day 21, ex-
plants were transferred to hormone-free medium. Sd" indicates the
presence of labelled and unlabelled spermidine in the medium.
day 21 day 30
Treatment ADC ODC ADC ODC
Control 169±20 161 ±20 128± 6 543± 92
MGBG 214±37 268±52 199± 18 478±118
CHA 466±39 429±57 571 ± 64 308± 25
MGBG+Sd* 798± 134 544± 33
CHA+Sd* 1459±172 370± 17
and CHA, while ODC was not affected (MGBG) or was
depressed (CHA).
The addition of spermidine to the inhibitor caused a fur-
ther strong enhancement of ADC activity both in MGBG
and CHA treatments while no changes (MGBG+spermi-
dine) or a decrease (CHA + spermidine) in ODC activity was
observed.
Discussion
Rhizogenesis was strongly inhibited in cultured tobacco
thin layers in the presence of MGBG and only slightly in the
presence of CHA. In the former, reversion of inhibition was
achieved by spermidine. The extent of inhibition was
slightly lower than that previously observed in the same
plant system using twice the concentration of the drugs (AI-
tamura et al., 1991); transfer of the cultures to hormone-free
medium on day 21 may also have affected this result.
In the presence of the drugs, rooting inhibition was accom-
panied, on day 21, by depletion of spermidine and putres-
cine, and on day 30, by depletion of spermidine and putres-
cine (MGBG) or depletion of free spermidine and accumula-
tion of bound spermidine and putrescine (CHA). The pre-
sent data are in agreement with the previously observed
CHA-induced changes in polyamine pattern, but are in con-
trast to the MGBG-induced spermidine and putrescine ac-
cumulation at the end of the culture period (Altamura et a1.,
1991). Thus, the absence of hormones in the last part of the
culture period seems to affect the action of MGBG but not
that ofCHA.
The inhibition of polyamine biosynthesis does not com-
pletely block the proliferative growth of the explants, but
highly disturbs it, depending on the drug and/or on the
polyamine depletion level, by inducing in some cells pheno-
mena of cell expansion (hypertrophy and/or elongation) and
inhibition of cell division (Bagni et al. , 1993). These pheno-
mena, provoked by difluoromethylornithine plus difluoro-
methylarginine (DFMO+DFMA), MGBG and CHA, are
reverted by the corresponding polyamine and could be ex-
plained on the basis of a possible block of the cells in the S-
phase due to polyamine depletion (Heby, 1981), as suggested
also by Berlin and Forche (1981) in DFMO-treated tobacco
cells. A pertubation of the cytoskeletal structures (microtu-
Rhiwgenesis regulation by polyamines 85
buIes and microfilaments) could also be involved m this
event.
In the present paper we also observed, mainly in the pres-
ence of MGBG, i.e. in the treatment that causes maximum
polyamine depletion, a high percentage of swollen explants
at the end of the culture period; this was probably due to the
presence of hypertrophic cells. The addition of spermidine
to MGBG strongly reverted explant hypertrophy.
Moreover, agood reversion of the inhibition of rhizogene-
sis was obtained in MGBG-treated explants when spermi-
dine was added at the time of its maximum depletion (day
21), but not in those treated with CHA. Spermidine-induced
reversion of the inhibition may have depended on the use of
a lower concentration of the drug than in the previous work
(Altamura et al., 1991), and on the hormone-free culture
conditions. In fact, in a previous paper in which spermidine
was added with MGBG (1 mM) at the onset of culture, a his-
tological analysis revealed the presence of about 10-12 root,
meristemoids per explant on day 30 versus 1- 2 in the
MGBG treatment (Altamura, unpublished observation).
Thus, the spermidine-induced reversion observed in the pre-
sent paper could have enhanced meristemoid development
into macroscopically visible root primordia, even though
further meristemoid induction cannot be excluded.
The different target enzymes of the drugs could account
for the observed discrepancy in affecting the rhizogenic pro-
cess. However, the reversion of rooting inhibition by sper-
midine in MGBG-treated explants, not observed previously
(Altamura et al., 1991), was associated with a strong accumu-
lation of free spermidine and TCA-soluble conjugated put-
rescine. The same occurred for CHA treatments where no
reversion of inhibition was observed.
The depletion in free and bound polyamine contents on
day 21 were comparable in MGBG and CHA treatments,
but in the presence of CHA, the further rhizogenic response
that was already defined on day 18 (see Table 1) did not
change substantially. On day 30, the accumulation of free
and bound putrescine due to CHA seems to be the most sig-
nificant difference in the polyamine pattern induced by the
drugs, and the possibility that the increase in putrescine titer
counteracts the effects of the decreased spermidine levels
must be taken into account, as also suggested for animal sys-
tems (Heby, 1981). Moreover, the fact that MGBG inhibits
S-adenosylmethionine decarboxylase (SAMDC) activity,
which represents a switching point between polyamine and
ethylene synthesis (Biondi et al., 1990), could explain the dif-
ferent rhizogenic response; in fact, in tomato leaf discs, inhi-
bition of SAMDC activity, leading to polyamine depletion
and enhanced ethylene synthesis, inhibited rhizogenesis (Co-
leman et al., 1980).
On day 21, the enhancement of both ADC and ODC acti-
vities by CHA is not in agreement with the lack of putres-
cine accumulation, while on day 30 the rise in ADC activity
is consistent with the increase in putrescine titers. It is al-
ways difficult to correlate the enzyme activities (ODC and
ADC) measured in vitro with the accumulation of their
product in vivo; furthermore, polyamine oxidation activities
should also be considered.
In the presence of spermidine both CHA and MGBG
caused strong increases in ADC activity (11- and 6-fold with
86 P. TORRIGIANI, M. M. ALTAMURA, S. SCARAMAGLI, F. CAPITANI, G. FALASCA, and N. BAGNI
respect to controls and 2.5- and 4-fold with respect to the in-
hibitor alone) consistent with putrescine accumulation.
MGBG is reported to inhibit spermidine accumulation
and morphogenic processes; for example, it reduced poly-
amine content and radicle growth in germinating Acer seeds
(Walker et al., 1985). It also completely inhibited somatic
embryogenesis in cultured carrot cells (Minocha et al., 1990;
1991) and strongly reduced rhizogenesis in stem explants of
Vigna radiata (Friedman et al., 1982). On the other hand,
MGBG depleted spermidine and putrescine titers and
lowered SAMDC activity, but did not affect the further de-
velopment of somatic embryos in Picea abies (Santanen and
Simola, 1992). Finally, MGBG administration to rats and
mice resulted in distinct ODC stimulation, attributable par-
tially to stabilization of the enzyme Ganne and Alhonen-
Hongisto, 1989). Thus, MGBG does or does not induce put-
rescine accumulation depending on the stage of the culture,
on the type of organ, as well as on the concentration used
and on the presence or obsence of diamine oxidase, which is
known to bind to MGBG in pea (Yanagisawa et al., 1981).
CHA is a well known inhibitor of embryogenesis in carrot
cell cultures and its effect is reversible by spermidine (Fien-
berg et al., 1984). In the same plant model, CHA depleted
spermidine and increased putrescine levels and ADC activ-
ity, but only delayed embryogenesis (Khan et al., 1991). In
cultured cotyledons of Pinus radiata, putrescine incorpora-
tion into spermidine and polyamine accumulation were
strongly reduced by CHA (Biondi et al., 1986; 1988). Fi-
nally, CHA caused scarce spermidine depletion but a rise in
putrescine titer and biosynthesis during the first cell cycle in
Helianthus tuberosus tuber explants (T orrigiani et al., 1987).
In rat brain tumor cells treated with CHA, spermidine fell
while putrescine increased (3.5-fold, Feuerstein et al., 1985).
Beyond this effect on polyamine titers, in HeLa tumor cells
ODC (the only putrescine biosynthetic enzyme in animal
cells) activity also increased and a 4-fold increase in the sta-
bility of the enzyme was found, which could be reversed by
exogenous spermidine (Mitchell et al., 1985).
Thus, CHA always depletes spermidine levels and very of-
ten increases those of putrescine. Nevertheless, the effects on
morphogenesis are less consistent and also seem to be linked
to the sensitivity of the plant model system (embryogenic
carrot cells, flower-forming thin layer explants) in addition
to a different drug uptake capacity (Biondi et al., 1986). The
putrescine accumulation often induced by CHA could be ex-
plained on the basis both of a lack of utilization for spermi-
dine synthesis and an increased biosynthetic activity (ODC
in animal cells and ADC in plant cells).
The interactions in the uptake process between MGBG, a
structural analog of spermidine, and spermidine (Pistocchi et
al., 1987) could explain not only the lower synthesis due to
its effect on SAMDC, but could also regulate the movement
of spermidine at the subcellular, cellular and tissue levels.
Moreover, in animal cells the two decarboxylases (ODC
and SAMDC) are rate-controlling enzymes as regards the
formation of spermidine and spermine, while aminopropyl-
transferases (spermidine- and spermine synthases) are not
Ganne and Alhonen-Hongisto, 1989). We can hypothesize
that also in plants such a correlation exists for ADC and
SAMDC, as our data seem to suggest. This would explain
the simultaneous depletion of putrescine and spermidine by
MGBG.
In the present paper there was some indication for a pos-
sible spermidine-to-putrescine pathway; in fact, spermidine
was incorporated to some extent into free and/or conjugated
putrescine in both CHA- and MGBG-treated explants. The
presence of an inverse metabolic link between spermidine
and putrescine, already reported in the literature (Bagni and
Speranza, 1977; Hartmann et al., 1988), needs to be better
demonstrated.
Acknowledgements
We wish to thank Mr. M. Cappella for technical support. This
research was supported by funds of Consiglio Nazionale delle Ri-
cerche (Italy), special project R.A.I.S.A., subproject n.2, paper n.
796.
References
fuTAMURA, M. M., P. TORRIGIANI, F. CAPITANI, S. SCARAMAGLI, and
N. BAGNI: De novo root formation in tobacco thin layers is af-
fected by inhibition of polyamine biosynthesis. J. Exp. Bot. 42,
1575-1582 (1991).
ALTAMURA, M. M., P. TORRIGIANI, G. FALASCA, P. ROSSINI, and N.
BAGNI: Morpho-functional gradients in superficial and deep tis-
sues along tobacco stem: polyamine levels, biosynthesis and oxi-
dation, and organogenesis in vitro. Submitted (1993).
BAGNI, N., M . M. ALTAMURA, S. BIONDI, M. MENGOLI, and P. TORRI-
GIANI: Polyamines and morphogenesis in normal and transgenic
plant cultures. In: ROUBELAKIS-ANGELAKIS, K. A. and M. TRAN
THANH VAN (eds.): Morphogenesis in Plants: Molecular Ap-
proaches, Plenum Press, New York, in press (1993).
BAGNI, N. and P. TORRIGIANI: Polyamines: a new class of growth
substances. In: KARSSEN, C. M., L. C. VAN LOON, and D. VREUG-
DENHIL (eds.): Progress in Plant Growth Regulation, Kluwer
Academic Publishers, Dordrecht, pp. 264-275 (1992).
BAGNI, N. and A. SPERANZA: Pathways of polyamine biosynthesis
during the growth of Helianthus tuberosus parenchymatic tissue.
In: KUDREV, T., I. IVANovA, and E. KARANOV (eds.): Plant
Growth Regulators, Publishing House of the Bulgarian Acad-
emy of Sciences, Sofia, pp. 75-78 (1977).
BERLIN, J. and E. FORCHE: DL-a-difluoromethylornithine causes en-
largment of cultured tobacco cells. Z. Pflanzenphysio!. 101,
277 -282 (1981).
BIONDI, S., N . BAGNI, and A. SANSOVINI: Dicyclohexylamine uptake
and effects on polyamine content in cultured cotyledons of ra-
diata pine. Physio!. Plant. 66, 41-45 (1986).
BIONDI, S., T. DIAz, I. IGLESIAS, G. GAMBERINI, and N. BAGNI: Poly-
amines and ethylene in relation to adventitious root formation
in Prunus avium shoot cultures. Physio!. Plant. 78, 474-483
(1990).
BIONDI, S., P. TORRIGIANI, A. SANSOVINI, and N. BAGNI: Inhibition
of polyamine biosynthesis by dicyclohexylamine in cultured
cotyledons of Pinus radiata. Physio!. Plant. 72,471-476 (1988).
BRADFORD, M. M.: A rapid and sensitive method for the quantifica-
tion of microgram quantities of protein utilizing the principle of
dye-binding. Anal. Biochem. 72,248-254 (1976).
COLEMAN, W. K., T . J. HUXTER, D. M. REID, and T. A. THORPE:
Ethylene as an endogenous inhibitor of root regeneration in to-
mato leaf discs cultured in vitro. Physio!. Plant. 48, 519-525
(1980).

FEUERSTEIN, B. G., D. F. DEEN, and L. J. MARTON: Effects of dicyclo-
hexylamine sulfate, a spermidine synthase inhibitor, in 9L rat
brain tumor cells. Cancer Res. 45, 4950-4954 (1985).
FIENBERG, A. A., J. H. CHOI, W. P. LUBICH, and Z. R. SUNG: Devel-
opmental regulation of polyamine metabolism in growth and
differentiation of carrot culture. Planta 162,532-539 (1984).
FRIEDMAN, R., A. ALTMAN, and U. BACHRACH: Polyamines and root
formation in mung bean hypocotyl cuttings. 1. Effect of exogen-
ous compounds and changes in endogenous polyamine content.
Plant Physiol. 70, 844-848 (1982).
GALSTON, A. W. and H. E. FLORES: Polyamines and plant morpho-
genesis. In: SLOCUM, R. D. and H. E. FLORES (eds.): Biochemistry
and Physiology of Polyamines in Plants, CRC Press, Boca Ra-
ton, Florida, pp. 175-186 (1991).
HARTMANN, T., H. SANDER, R. ADOLPH, and G. TOPPEL: Metabolic
links between the biosynthesis of pyrrolizidine alkaloids and
polyamines in root cultures of Senecio vulgaris. Planta 175, 82-
90 (1988).
HEBY, 0.: Role of polyamines in the control of cell proliferation
and differentiation. Differentiation 19,1-20 (1981).
KHAN, A. J. and S. C. MINOCHA: Polyamines and somatic embryo-
genesis in carrot. II. The effects of cyclohexylammonium phos-
phate. J. Plant Physiol. 137, 446-452 (1991).
JANNE, J. and L. ALHONEN-HONGISTO: Inhibitors of polyamine bio-
synthesis as therapeutic agents. In: BACHARACH, U. and Y. M.
HEIMER (eds.): The Physiology of Polyamines, Vol. II, CRC
Press, Boca Raton, Florida, pp. 251- 286 (1989).
MINOCHA, S. c., R. MINOCHA, and A. KOMAMINE: Effects of poly-
amine biosynthesis inhibitors on S-adenosylmethionine synthe-
tase and S-adenosylmethionine decarboxylase activities in carrot
cell cultures. Plant Physiol. Biochem. 29, 231-237 (1991).
Rhizogenesis regulation by polyamines 87
MINOCHA, S. C., C. A. ROBIE, A. J. KAHN, N. PAPA, A.I. SAMUELSEN,
and R. MINOCHA: Polyamine and ethylene biosynthesis in rela-
tion to somatic embryogenesis in carrot (Daucus carota L.) cell
cultures. In: FLORES, H. E., R. N. ARTECA, and J. C. SHANNON
(eds.): Polyamines and Ethylene: Biochemistry, Physiology, and
Interactions, American Society of Plant Physiologists, Rock-
ville, Maryland, pp. 339-342 (1990).
MITCHELL, J. L. A., D. W. MAHAN, P. P. MCCANN, and P. QASBA:
Dicyclohexylamine effects on HTC cell polyamine content and
ornithine decarboxylase activity. Biochim. Biophys. Acta 840,
309-315 (1985).
PISTOCCHI, R., N. BAGNI, and J. A. CREUS: Polyamine uptake in car-
rot cell cultures. Plant Physiol. 84, 374-380 (1987).
SANTANEN, A. and L. K. SIMOLA: Changes in polyamine metabolism
during somatic embryogenesis in Picea abies. J. Plant Physiol.
140,475-480 (1992).
TORRIGIANI, P., M. M. ALTAMURA, F. CAPITANI, D. SERAFINI-FRACAS-
SINI, and N. BAGNI: De novo root formation in thin cell layers of
tobacco: changes in free and bound polyamines. Physiol. Plant.
77,294-301 (1989).
TORRIGIANI, P., D. SERAFINI-FRACASSINI, and N. BAGNI: Polyamine
biosynthesis and effect of dicyclohexylamine during the cell cy-
cle of Helianthus tuberosus tuber. Plant Physiol. 84, 148 -152
(1987).
WALKER, M. A., D. R. ROBERTS, C. Y. SHIH, and E. B. DUMBROFF: A
requirement for polyamines during the cell division phase of
radicle emergence in seeds of Acer saccharum. Plant Cell Physiol.
26, 967 -971 (1985).
YANAGISAWA, H., E. HIRASAWA, and Y. SUZUKI: Purification and pro-
perties of diamine oxidase from pea epicotyls. Phytochemistry
20,2105-2108 (1981).