Download Regulation by Polyamines of Ornithine

Regulation by Polyamines of Ornithine Decarboxylase
Activity and Cell Division in the Unicellular Green Alga
Chlamydomonas reinhardtii1
Christine Theiss, Peter Bohley, and Jürgen Voigt*
Physiologisch-Chemisches Institut, Universität Tübingen, Hoppe-Seyler-Stra␤e 4, D–72076 Tübingen,
Germany
Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green
alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine)
is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is
the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was
preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine
levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC
activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when
applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this
growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or
putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the
findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be
prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after
beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via
protein biosynthesis.
Polyamines are ubiquitous cell components essential for normal growth of both eukaryotic and prokaryotic cells (Tabor and Tabor, 1984; Marton and
Pegg, 1995). In higher plants, polyamines also influence developmental processes and play an important
role in the response to abiotic stress (Galston et al.,
1997). The three commonly occurring polyamines
(putrescine, spermidine, and spermine) are synthesized from Orn and/or Arg, putrescine being the first
polyamine in these biosynthetic pathways (Slocum,
1991). Spermidine and spermine are generated from
putrescine by the addition of aminopropyl groups
derived from decarboxylated S-adenosyl Met (Slocum, 1991). The rate-limiting step in the formation of
putrescine in animals and most fungi is the decarboxylation of Orn by Orn decarboxylase (ODC; for
references, see Marton and Pegg, 1995). In some
plants, putrescine is also synthesized via the decarboxylation of Arg by Arg decarboxylase and subsequent degradation of the generated agmatine (Pegg,
1986; Minocha and Minocha, 1995; Kumar et al., 1997;
Walden et al., 1997; Andersen et al., 1998). As previously reported, putrescine formation in the unicellu1
This work was supported by the Deutsche Forschungsgemeinschaft (grant no. Vo 327/9).
* Corresponding author; e-mail juergen.voigt@uni-tuebingen.
de; fax 49 –7071–295009.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010896.
1470
lar green alga Chlamydomonas reinhardtii, however, is
controlled by ODC rather than by Arg decarboxylase
activity (Voigt et al., 2000a).
ODC activity is regulated both at the translational
level (Tabor and Tabor, 1984; Marton and Pegg, 1995)
and via controlled, ATP-dependent proteolysis by
the 26S proteasome, at least in mammalian and yeast
(Saccharomyces cerevisiae) cells (Bercovich et al., 1989;
Mamroud-Kidron and Kahana, 1994). ODC is an enzyme with rather short half-life, varying between 30
and 120 min in all eukaryotic organisms studied so
far (Voigt et al., 2000a) with the exception of Trypanosoma brucei (t1/2 ⫽ 6 h; Phillips et al., 1987). In synchronized cultures of the unicellular green alga C.
reinhardtii, a 2.5- to 3-fold increase of the ODC halflife was observed during transition to the cell division phase, accompanied by a 3-fold increase of ODC
activity (Voigt and Bohley, 2000). For mammalian
cells, it has been reported that degradation of ODC is
not mediated by ubiquitination (Bercovich et al.,
1989), but by binding of ODC monomers to an inhibitory protein named antizyme (Murakami and Hayashi, 1985; Hayashi and Murakami, 1995; Hayashi et
al., 1996). At least two different pathways for degradation of ODC in mammalian cells are known as yet,
a constitutive and a polyamine-dependent pathway
(Li and Coffino, 1993). The C-terminal domain is
necessary in both cases and sufficient to make an
ODC molecule constitutively unstable. Surface hydrophobicity and PEST sequences (sequences with
Plant Physiology, Downloaded
April 2002, Vol.
from128,
on June
pp. 1470–1479,
16, 2017 - Published
www.plantphysiol.org
by www.plantphysiol.org
© 2002 American Society of Plant Biologists
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Polyamines Regulate Cell Division in Chlamydomonas reinhardtii
high proportions of Pro, Glu/Asp, Ser, and Thr and
lacking basic amino acid residues) are actually considered as signal structures for the rapid and selective degradation of specific proteins by the proteasome (Bohley, 1996; Rechsteiner and Rogers, 1996).
For several years, a PEST sequence in the C-terminal
domain of mammalian ODCs (PEST2) was assumed
to be responsible for the rapid degradation of the
ODC subunits because this particular PEST sequence
is missing in the long-living ODC of T. brucei (Phillips
et al., 1987; Rechsteiner and Rogers, 1996). However,
stabilization of mammalian ODC was achieved by
deletion of the C-terminal pentapeptide Ala Arg Ile
Asn Val, whereas deletions affecting the neighboring
PEST2 sequence did not increase ODC stability
(Ghoda et al., 1992; Li and Coffino, 1992, 1993). Although ODC is also a short-living enzyme in plant
cells (Hiatt et al., 1986; Voigt and Bohley, 2000; Voigt
et al., 2000a), there are still no published data concerning the reason for its rapid degradation in plant
cells. The control of ODC degradation by the intracellular polyamine level and the antizyme level (Murakami and Hayashi, 1985; Hayashi and Murakami,
1995; Hayashi et al., 1996) is proposed to assure
homeostatic regulation of both the ODC activity and
the polyamine concentrations in mammalian cells.
However, elevated polyamine levels have been
found in proliferating plant and mammalian cells as
well as in cancer cells (Hibshoosh et al., 1991; Auvinen et al., 1992; Moshier et al., 1993; Daoudi and
Biondi, 1995; Marton and Pegg, 1995; Ben Hayyim et
al., 1996; Fowler et al., 1996; Cvikrova et al., 1999). A
rapid increase in ODC activity was measured when
growth-arrested mammalian or plant cells were
transferred to fresh culture medium enabling cell
proliferation (Manzella et al., 1991; Fowler et al.,
1996; Graff et al., 1997). This increase in ODC activity
was found to be mediated by an increased translation
of preexisting ODC mRNA (Manzella et al., 1991;
Graff et al., 1997). A rapid up-regulation of ODC activity was also observed when dark-adapted (starved)
C. reinhardtii cells were transferred to the light (Voigt
et al., 2000a). This light-induced increase in ODC activity was abolished by the PSII inhibitor 3-(3,4dichlorophenyl)-1,1-dimethylurea and could be prevented by inhibition of protein biosynthesis, but not
by inhibition of RNA synthesis (Voigt et al., 2000a).
An increase in ODC activity was also observed
when (partially) synchronized mammalian cells entered the cell division phase (Koza and Herbst 1992;
Fredlund et al., 1995). However, the mechanism of
this putative cell cycle control of ODC is completely
unknown. Unicellular green algae like C. reinhardtii
and Scenedesmus obliquus can be easily synchronized
by cultivation under a constant light-dark regime
(Voigt and Münzner, 1987; Krupinska and Humbeck,
1994). Analyses of synchronized cultures of Scenedesmus revealed that the polyamine levels increased
during the growth period and decreased after the cell
Plant Physiol. Vol. 128, 2002
division phase (Kotzabasis and Senger, 1994). In synchronized cultures of the unicellular green alga C.
reinhardtii, a 3-fold increase in ODC activity was observed during the transition to the cell division phase
that correlated with a 3-fold increase of the ODC
half-life (Voigt and Bohley 2000). Therefore, we have
investigated the effects of polyamines on ODC activity and cell cycle progression in synchronized cultures of C. reinhardtii.
RESULTS
Cell Cycle-Dependent Alteration of the
Polyamine Levels
In synchronized C. reinhardtii cultures growing under a 14-h-light/10-h-dark regime, the intracellular
levels of putrescine and spermidine strongly increased during the light phase (growth period) as
shown in Table I. A 10-fold increase of putrescine
and 20-fold increase of spermidine was measured
15 h after onset of illumination (Table I). The spermidine level was much lower (3%–10% of total polyamines) than the putrescine level (90%–97% of total
polyamines; Table I). Spermine could not be detected. Therefore, the question arose whether or not
this cell cycle-dependent alteration of the polyamine
level affects cell cycle progression.
Down-Regulation by Polyamines of ODC Activity in
C. reinhardtii Cells
Because ODC is the key enzyme of polyamine biosynthesis in C. reinhardtii (Voigt et al., 2000a), we
have investigated the response of this particular enzyme to different exogenous concentrations of putrescine, spermidine, and spermine, respectively.
When added 8 h after beginning of the light period,
all these commonly occurring polyamines caused a
decrease in ODC activity after 5 h (Table II). In the
case of putrescine, however, a considerably higher
Table I. Cell cycle-dependent changes of the levels of intracellular
polyamines
Cultures of the wall-deficient strain C. reinhardtii cw-15 were
synchronized by growth under a constant 14-h-light/10-h-dark regime. Cells were harvested at the indicated time intervals after onset
of illumination and analyzed for free polyamines as described in
“Materials and Methods.” Mean values of three individual experiments ⫾ SD are given.
Time after Onset of
Illumination
Putrescine
a
Spermine
9
h
0
5
10
15
20
Spermidine
nmol/10 cells
14 ⫾ 3
32 ⫾ 5
91 ⫾ 11
168 ⫾ 18
135 ⫾ 16
0.4 ⫾ 0.05
1.1 ⫾ 0.22
3.3 ⫾ 0.5
7.8 ⫾ 1.1
6.4 ⫾ 1.2
nda
nd
nd
nd
nd
nd, Not detected.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1471
Theiss et al.
Table II. Effects of exogenous polyamines on ODC activity in
C. reinhardtii cells
Cultures of the wall-deficient strain C. reinhardtii cw-15 (5 L) were
grown under a 14-h light/10-h dark regime to a cell density of 0.5 to
1.0 ⫻ 106 cells mL⫺1. At the day of the experiment, 200-mL aliquots
were taken from these cultures 8 h after onset of illumination and
further incubated in the light in the presence of different concentrations of putrescine, spermidine, and spermine, respectively. After 5 h,
cells were harvested by centrifugation and cell lysates prepared and
analyzed for ODC activity and protein as described in “Materials and
Methods.” Specific ODC activities are shown as percentage of the
maximal activity of each experiment. Mean values of five separate
experiments ⫾ SD are given. P values were calculated by Student’s
t test.
Polyamine
Concentration
mmol L
–
Putrescine
Putrescine
Putrescine
Putrescine
Putrescine
Putrescine
Spermidine
Spermidine
Spermidine
Spermidine
Spermine
Spermine
Spermine
Spermine
–
0.05
0.10
0.20
0.50
0.75
1.00
0.02
0.05
0.07
0.10
0.02
0.05
0.07
0.10
⫺1
ODC Activity
Student’s t Test
%
100.0
98.0 ⫾ 3.4
97.1 ⫾ 3.2
91.4 ⫾ 7.6
82.5 ⫾ 10.2
69.8 ⫾ 13.1
62.4 ⫾ 6.9
78.1 ⫾ 16.7
61.3 ⫾ 12.3
50.9 ⫾ 10.5
44.7 ⫾ 5.9
79.5 ⫾ 18.4
67.1 ⫾ 16.3
54.7 ⫾ 9.8
48.2 ⫾ 5.3
–
Nonsignificant
Nonsignificant
P ⬍ 0.05
P ⬍ 0.005
P ⬍ 0.0005
P ⬍ 0.0001
P ⬍ 0.005
P ⬍ 0.0005
P ⬍ 0.0001
P ⬍ 0.0001
P ⬍ 0.025
P ⬍ 0.00025
P ⬍ 0.0001
P ⬍ 0.0001
exogenous concentration was required for a significant down-regulation of ODC activity than for spermidine and spermine, respectively (Table II). A decrease in ODC activity by 30% to 35% was observed
when putrescine was applied at a concentration of 1
mmol L⫺1. Spermidine and spermine, however,
caused the same effect at concentrations of about 0.05
mmol L⫺1 (Table II). Cytotoxic effects were observed
when putrescine was applied at concentrations above
1.5 mmol L⫺1 (data not shown). No cytotoxic effects
were observed in the presence of up to 0.3 mmol L⫺1
of spermidine or spermine. In the following experiments, therefore, the effects of putrescine were studied at an exogenous concentration of 1 mmol L⫺1,
and the effects of spermidine and spermine at concentrations of 0.1 mmol L⫺1.
Effects of Exogenous Polyamines on Cell CycleDependent Alteration of ODC Activity and
Cell Division
In synchronized cultures of the unicellular green
alga C. reinhardtii, a 4-fold increase in ODC activity
was measured during the transition from the cell
enlargement to the cell division phase (Fig. 1D) preceding the increase in aphidicoline-sensitive DNA
polymerase activity (S-phase; Fig. 1C) and the accumulation of dividing cells (Fig. 1B). This cell cycle1472
dependent increase in ODC activity was abolished
when exogenous polyamines were added 7 to 8 h
after beginning of the light period (growth phase;
Fig. 1D). After addition of spermine, a transient
decrease of ODC activity was measured (Fig. 1D).
To minimize effects on cell growth, polyamines
were added 7 to 8 h after beginning of the light
period when the cells had already doubled their cell
mass and therefore were able to divide (Voigt and
Münzner, 1987, 1989). Addition of putrescine or
Figure 1. Effects of polyamines on ODC activity and cell divisions in
synchronized C. reinhardtii cultures. Cultures of the wall-deficient C.
reinhardtii strain cw-15 were synchronized by growth under a constant light-dark regime of 14 h of light and 10 h of darkness for 4 d.
On the day before the experiment, the cultures were divided and the
subcultures diluted with fresh culture medium. Putrescine (final concentration 1 mmol L⫺1), spermidine (final concentration 0.1 mmol
L⫺1), and spermine (final concentration 0.1 mmol L⫺1), respectively,
were added 8 h after beginning of the light period (growth phase).
The cultures were analyzed for cell densities (A) and dividing cells (B)
at the time periods after onset of illumination indicated in the figure.
To determine the DNA polymerase-␣ (C) and ODC activities (D),
aliquots (400 mL) of the cultures were harvested and cell lysates and
nuclei were prepared and assayed for ODC activities, protein concentrations, and DNA polymerase-␣ activity as described in “Materials and Methods.” Aphidicolin-sensitive DNA polymerase ␣ activities are expressed in 104 cpm incorporated by 5 ⫻ 106 nuclei. ODC
activities (D) are expressed in percent of maximal specific activity of
each separate experiment, which varied between 1,150 and 2,180
␮-units ODC mg protein⫺1. Values are means ⫾ SD of four separate
experiments. ‚, Putrescine; , spermidine; E, spermine; 〫, control.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Polyamines Regulate Cell Division in Chlamydomonas reinhardtii
Table III. Effect of exogenous spermine on the intracellular polyamine level
Cultures of the wall-deficient strain C. reinhardtii cw-15 were synchronized by growth under a
constant 14-h-light/10-h-dark regime. Spermine was added to a final concentration of 100 ␮mol L⫺1 8 h
after beginning of the light period. Cells were harvested at the indicated time intervals after onset of
illumination and analyzed for free polyamines as described in “Materials and Methods.” Mean values
of three individual experiments ⫾ SD are given.
Time after Onset of
Illumination
Putrescine
Spermidine
a
Spermine
Putrescine
Spermidine
Spermine
2.0 ⫾ 0.3
1.2 ⫾ 0.4
3.7 ⫾ 0.8
7.2 ⫾ 1.5
16 ⫾ 3.6
22 ⫾ 2.9
99 ⫾ 10.8
109 ⫾ 12.1
nmol/109 cells
h
9
12
15
18
⫹Spermine
Control
88 ⫾ 9
106 ⫾ 11
135 ⫾ 14
166 ⫾ 16
2.1 ⫾ 0.2
4.8 ⫾ 0.5
7.4 ⫾ 1.1
13 ⫾ 1.5
nda
nd
nd
nd
84 ⫾ 9
48 ⫾ 7
76 ⫾ 11
63 ⫾ 14
nd, Not detected.
spermidine did not affect the increase in cell density
(Fig. 1A) and the timing of cell division (Fig. 1B).
Spermine, however, caused a cell cycle arrest under
the same conditions (Fig. 1, A and B). To investigate
whether or not the transition from G1 to S phase was
affected by exogenous polyamines, crude nuclei were
analyzed for the activity of the aphidicoline-sensitive
DNA polymerase ␣ (Fig. 1C). Aphidicoline-sensitive
DNA polymerase-␣ activity was detected in nuclei
from untreated and putrescine- or spermidinetreated cells, but not in the nuclei from sperminetreated cells (Fig. 1C). Furthermore, cells in
spermine-treated cultures retained their motility,
whereas in untreated and putrescine- or spermidinetreated cultures, cells lost their motility before cell
division (data not shown; for references, see Harris,
1989). These findings indicate that spermine affects
the transition from the G1 to the S phase. In this
context, it is important to know that under optimal
growth conditions, C. reinhardtii cells are able to multiply their cell mass within the light phase (growth
period, G1 phase). During the subsequent cell division phase, these “mother cells” divide several times
without additional G1 phases and with extremely
short G2 phases that cannot be detected under normal experimental conditions (Harris, 1989).
The Spermine-Induced Cell Cycle Arrest Is
Due to a Lack of Spermidine
Because exogenous polyamines affected ODC activity (Table II; Fig. 1D), the spermine-induced cell
cycle arrest might be caused by a lack of putrescine
and/or spermidine. Comparative analyses of the intracellular polyamine levels in spermine-treated and
-untreated cultures revealed that spermine caused a
decrease in the putrescine and spermidine levels and
a reduced cell cycle-dependent increase of both polyamines (Table III). For this reason, we have investigated whether or not the spermine-induced cell cycle
arrest can be overcome by a subsequent addition of
putrescine or spermidine. Cell divisions were induced in spermine-treated cells by addition of sperPlant Physiol. Vol. 128, 2002
midine and to a lower extend also by addition of
putrescine (Fig. 2). Furthermore, we studied the effects of inhibitors of spermidine synthesis on cell
division in synchronously growing C. reinhardtii cultures. The inhibitors were applied 8 h after beginning
Figure 2. Spermine-induced cell cycle arrest is overcome by addition
of putrescine or spermidine. On the day before the experiment, synchronized cultures of the wall-deficient C. reinhardtii strain cw-15
were divided and the two subcultures diluted with fresh culture medium to a final cell density of 0.5 ⫻ 106 cells mL⫺1 and further
incubated under a 14-h-light/10-h-dark regime. Spermine (final concentration 0.1 mmol L⫺1) was added to one of these cultures 8 h after
beginning of the subsequent light period (growth phase). After 4 h, the
spermine-treated culture was divided into three subcultures. Putrescine (final concentration 1 mmol L⫺1) and spermidine (final concentration 0.1 mmol L⫺1), respectively, were added to two of these
subcultures (indicated by arrow). The cultures were analyzed for dividing cells (A) and cell density (B) at the time periods after onset of
illumination indicated in the figure. Values are means ⫾ SD of five
separate experiments. 〫, Control; E, spermine; ‚, spermine ⫹ putrescine; , spermine ⫹ spermidine.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1473
Theiss et al.
ied (Fig. 3, B and C). Cell divisions were largely
abolished when the SAMDC inhibitor MGBG was
added to a final concentration of 200 ␮mol L⫺1 (Fig.
3B). At a concentration of 50 ␮mol L⫺1, MGBG
caused a slight increase of dividing cells and a delay
in the increase of the cell density (Fig. 3B). Addition
of the ODC inhibitor DFMO at a concentration where
a quantitative inhibition of ODC was measured after
5 h (Table IV), however, did not abolish cell divisions, but caused an early accumulation of dividing
cells (Fig. 3C). Cell density increased, however, at the
same time as in the untreated cultures (Fig. 3, A and
C). In the presence of both DFMO and MGBG, cell
divisions were abolished (Fig. 3C). Significantly increased ODC activities were measured 5 h after addition of the SAMDC inhibitor MGBG or of the spermidine synthase inhibitor 4-Me-CHA (Table IV) as
already reported for mammalian cells (Marton and
Pegg, 1995).
The Spermine-Induced Cell Cycle Arrest Is Not
Due to a Decreased Rate of Protein Synthesis
Figure 3. Effects of inhibitors of the polyamine metabolism on cell
divisions in synchronized C. reinhardtii cultures. Cultures of the
wall-deficient C. reinhardtii strain cw-15 were synchronized by
growth under a constant light-dark regime of 14 h of light and 10 h
of darkness for 4 d. On the day of the experiment, the cultures were
divided and different inhibitors of the polyamine metabolism were
added to the subcultures 8 h after beginning of the light period. The
cultures were analyzed for cell densities (solid lines) and dividing
cells (dashed lines) at the time periods after onset of illumination
indicated in the figure. A, Untreated control (〫) and addition of the
spermidine synthase inhibitor 4-trans-methyl-cyclohexyl-amine (1
mmol L⫺1; f); B, addition of the S-adenosyl-Met decarboxylase
(SAMDC) inhibitor methylglyoxal-bis-(guanyl-hydrazone) (MGBG) to
final concentrations of 50 ␮mol L⫺1 (‚) and 200 ␮mol L⫺1 (),
respectively; C, addition of the ODC inhibitor difluoromethylornithine (DFMO; 2 mmol L⫺1) in the absence (ƒ) or presence (Œ) of 200
␮mol L⫺1 of the SAMDC inhibitor MGBG.
of the light period when most of the cells had doubled their cell mass (Voigt and Münzner, 1987, 1989).
Cell divisions were abolished by addition of
4-methyl-cyclohexylamine (4-Me-CHA; Fig. 3A), an
inhibitor of spermidine synthase (Marton and Pegg,
1995). Because spermidine is generated from putrescine and decarboxylated S-adenosyl-Met, which
are formed by ODC and SAMDC, respectively, the
effects of inhibitors of these enzymes were also stud1474
Because spermidine is required for the activation of
eIF-5A (Jakus et al., 1993), we have investigated
whether or not the spermine-induced cell cycle arrest
mediated by a decreased spermidine level is caused
by a down-regulation of protein synthesis. To this
end, aliquots were taken from synchronized cultures
at different time intervals after onset of illumination,
pulse labeled with [3H]Arg, and subsequently analyzed for radioactively labeled protein. In untreated
cultures, incorporation of [3H]Arg into protein increased during the growth period (light phase) up to
13 h after onset of illumination (Fig. 4) and declined
Table IV. Inhibitors of spermidine synthesis affect ODC activity
Cultures of the wall-deficient strain C. reinhardtii cw-15 (2 L) were
synchronized by growth under a 14-h-light/10-h-dark regime. On the
day of the experiment, 200-mL aliquots were taken from these cultures 8 h after beginning of the light period and further incubated in
the light after addition of inhibitors of spermidine synthesis: DFMO,
inhibitor of ODC; MGBG, inhibitor of SAMDC; and 4-Me-CHA,
inhibitor of spermidine synthase. After 5 h, cells were harvested by
centrifugation and cell lysates prepared and analyzed for ODC activity and protein as described in “Materials and Methods.” Specific
ODC activities are shown as percentage of the control (ODC activity
in untreated cells). Mean values of five separate experiments ⫾ SD are
given. P values were calculated by Student’s t test.
Inhibitor
–
DFMO
MGBG
MGBG
DFMO
⫹
MGBG
4-MeCHA
Concentration
ODC Activity
mM
%
–
2
0.05
0.20
2
0.20
100
2 ⫾ 3.2
380 ⫾ 110
700 ⫾ 130
–
P ⬍ 0.0001
P ⬍ 0.0001
P ⬍ 0.0001
1.9 ⫾ 0.3
P ⬍ 0.0001
2
210 ⫾ 65
P ⬍ 0.00025
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Student’s t Test
Plant Physiol. Vol. 128, 2002
Polyamines Regulate Cell Division in Chlamydomonas reinhardtii
Figure 4. Effects of spermine and spermidine on cell cycle-dependent variation of the incorporation of [3H]Arg into protein.
Cultures of the wall-deficient strain C. reinhardtii cw-15 were synchronized by growth under a constant 14-h-light/10-h-dark
regime. At the day before the experiment, the cultures were divided and diluted with fresh culture medium to a density of
0.5 ⫻ 106 cell mL⫺1. Spermine and spermidine were added 8 and 13 h, respectively, after beginning of the light period (final
concentrations: 0.1 mmol L⫺1). Aliquots of 1 mL were taken at the indicated time intervals after onset of illumination and
pulse labeled with 370 kBq of [3H]Arg for 30 min. After pulse labeling, the cells were harvested, washed, and analyzed for
radioactively labeled protein as described in “Materials and Methods.” Data are expressed in dpm [3H]Arg incorporated into
protein per 106 cells. Mean values of four separate experiments ⫾ SD are given. 〫, Control culture; E, culture treated with
spermine (0.1 mmol L⫺1) 8 h after beginning of the light period; , culture treated with spermine (0.1 mmol L⫺1) 8 h after
beginning of the light period and with spermidine (0.1 mmol L⫺1) after 13 h.
subsequently during cell division (Figs. 1 and 4).
Addition of spermine 8 h after beginning of the light
period did not cause a decrease in protein biosynthesis (Fig. 4). Instead, the decrease in the incorporation
of [3H]Arg into protein observed in the untreated
cultures between 15 and 21 h after onset of illumination was not observed in the presence of spermine
unless spermidine was additionally applied 13 h after beginning of the light phase (Fig. 4).
DISCUSSION
In C. reinhardtii cells, an almost constant putrescine:
spermidine ratio of about 10:1 was found at all cell
cycle stages (Table I). Spermine could not be detected
in C. reinhardtii cells (Table I). In synchronously
growing C. reinhardtii cells, a 10- and a 20-fold increase, respectively, in the putrescine and spermidine
levels was observed during the light period the maximal level being reached 15 h after onset of illumination (Table I) as already reported for the green alga
S. obliquus (Kotzabasis and Senger, 1994). The increase in the polyamine level was accompanied by a
4-fold increase in the activity of ODC (Fig. 1D), the
key enzyme of polyamine synthesis in C. reinhardtii
(Voigt et al., 2000a). This cell cycle-dependent increase in ODC activity preceded the transition to the
cell division phase (Fig. 1, B and C) and was found to
be caused by an increased ODC half-life (Voigt and
Bohley, 2000). Therefore, the question arose whether
Plant Physiol. Vol. 128, 2002
or not this increase in ODC activity and polyamine
levels has any significance for cell division. Increased
ODC activities and polyamine levels were also observed in proliferating mammalian and higher plant
cells as compared with resting cells (Manzella et al.,
1991; Daoudi and Biondi, 1995; Marton and Pegg,
1995; Ben Hayyim et al., 1996; El Ghachtouli et al.,
1996; Fowler et al., 1996; Graff et al., 1997; Chattopadhyay and Ghosh, 1998; Cvikrova et al., 1999).
Furthermore, ODC activities and polyamine levels
were found to be elevated in cancer cells (Hibshoosh
et al., 1991; Auvinen et al., 1992; Moshier et al., 1993;
Marton and Pegg, 1995). As previously reported, inhibition of ODC not only caused a decrease in the
polyamine levels, but also a decreased rate of cell
divisions (Koza and Herbst, 1992; Fredlund et al.,
1995). On the other hand, it has been shown that in
eukaryotic cells, spermidine is required for the activation of initiation factor eIF-5A (Jakus et al., 1993)
and, therefore, necessary for protein biosynthesis and
cell growth. For this reason, the published data indicating a correlation between polyamine level and cell
proliferation (Hibshoosh et al., 1991; Auvinen et al.,
1992; Moshier et al., 1993; Marton and Pegg, 1995)
and the findings that inhibition of of polyamine synthesis impaired division of eukaryotic cells (Koza
and Herbst, 1992; Fredlund et al., 1995) could be
referred to as a dependence of protein biosynthesis
on the spermidine level. Thus, assumptions that
polyamines might be directly involved in the regula-
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1475
Theiss et al.
tion of cell division (Koza and Herbst 1992; Fredlund
et al., 1995) have not, up to now, been corroborated
by satisfying experimental proofs.
Unicellular green algae like C. reinhardtii are particularly suitable for such studies because they can be
easily synchronized by growth under a constant lightdark regime (Surzycki, 1971; Voigt and Münzner,
1987; Krupinska and Humbeck, 1994). Furthermore,
they do not enter the cell division phase upon doubling their cell mass. Under optimal growth conditions, C. reinhardtii cells can multiply their cell mass
during the light phase (growth period, G1 phase; for
references, see Schlösser, 1966; Mihara and Hase,
1971; Voigt and Münzner, 1987; Harris, 1989). During
the subsequent cell division phase that normally occurs during the dark period unless the cells have
reached a strain-specific maximal size already during
the light phase (Voigt and Münzner, 1987), these
“mother cells” undergo several cell divisions without
additional G1 phases and without measurable G2
phases (Harris, 1989). Direct effects on cell division of
compounds influencing both cell growth and cell
division can be studied in this experimental system
when applied after most of the cells have doubled
their cell mass and, therefore, attained the commitment to divide (e.g. 7–8 h after onset of illumination;
for references, see Voigt and Münzner, 1989). For this
reason, we have investigated the influence of both
exogenous polyamines and inhibitors of polyamine
metabolism on ODC activity and cell divisions in
synchronized C. reinhardtii cultures when added 7 to
8 h after beginning of the light period. Addition of
spermine, but not of putrescine or spermidine, inhibited the transition to the cell division phase (Fig. 1, B
and C). In untreated C. reinhardtii cultures, the transition to the S-phase (Fig. 1C) was preceded by a
4-fold increase in ODC activity (Fig. 1D). Addition of
polyamines prevented this up-regulation of ODC activity (Fig. 1D), indicating that the spermine-induced
cell cycle arrest (Fig. 1, A–C) might be due to a lack
of putrescine and/or spermidine. This conclusion
was corroborated by our observations that the levels
of putrescine and spermidine were decreased after
addition of spermine (Table III) and that the
spermine-induced cell cycle arrest was overcome by
subsequent addition of spermidine or, to a lesser
extent, also by addition of putrescine (Fig. 2). Furthermore, cell divisions could also be prevented by
addition of inhibitors of spermidine synthesis (Fig. 3)
with the exception of the ODC inhibitor DFMO. Because the ODC activity was shown to be completely
inhibited under these experimental conditions (Table
IV), the putrescine level must have been high enough
for the formation of amounts of spermidine sufficient
for the transition to the cell division phase under
these conditions. On the other hand, it has been
shown that inhibition of ODC by DFMO causes an
increase in SAMDC activity, thus resulting in an
1476
increased formation of spermidine (Marton and
Pegg, 1995).
The question arose whether the lack of spermidine
affects cell division directly or via a decrease in the
rate of protein biosynthesis caused by a decreased
spermidine-dependent activation of eIF-5A (Jakus et
al., 1993). As shown in Figure 4, no decrease in protein synthesis could be observed after addition of
spermine under conditions where spermine caused a
cell cycle arrest (Figs. 1 and 2). Addition of spermine
under these experimental conditions prevented the
decrease in protein biosynthesis normally observed
when the cells entered the division phase (Fig. 4; for
references, compare with Voigt et al., 2000b). This
finding was in agreement with the observation that
the loss of cell motility that accompanies the transition to the cell division phase was also prevented by
treatment with spermine (data not shown). In accordance, a decrease in protein biosynthesis (Fig. 4) and
a loss of cell motility (data not shown) were observed
when spermidine was added to cultures that were
cell cycle arrested by spermine treatment. These findings indicate that, at least in the case of C. reinhardtii,
spermidine is required for the transition from G1 to
the S phase and that, under our experimental conditions, spermidine does not affect cell division by its
effects on the activiation of eIF-5A (Jakus et al., 1993)
and, therefore, via protein synthesis and cell growth.
When added at the beginning of the light period,
however, polyamines caused an increase in protein
biosyntheis (data not shown) that can be explained
by effects on the activation of eIF-5A (Jakus et al.,
1993).
MATERIALS AND METHODS
Strains and Growth Conditions
The cell wall-deficient strain CW15 of Chlamydomonas
reinhardtii (Davies and Plaskitt, 1971) was obtained from
the Sammlung von Algenkulturen at the University of
Göttingen (Germany). Cells were grown at 24°C under a
photon fluence rate of 40 ␮mol m⫺2 s⫺1 in a high-salt
medium supplemented with 0.2% (w/v) sodium acetate as
described previously (Voigt and Münzner, 1987). Synchronized cultures were obtained by light-dark cycling under a
14-h-light/10-h-dark regime (Voigt and Münzner, 1987).
Cell concentrations were determined by duplicate hemocytometer counting.
Preparation of Cell Lysates
Cells were harvested by centrifugation at 6,000g for 10
min at 4°C. All subsequent steps were perfomed at 0°C to
4°C. The cells were washed with and resuspended to a final
cell densitity of 0.5 to 1 ⫻ 109 cells mL⫺1 in ice-cold
homogenization buffer A consisting of 25 mmol L⫺1 TrisHCl, pH 7.0, 2 mmol L⫺1 dithiothreitol, and 0.1 mmol L⫺1
EDTA. After addition of the protease inhibitors phenylmethylsulfonyl fluoride (final concentration: 0.1 mmol L⫺1)
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Polyamines Regulate Cell Division in Chlamydomonas reinhardtii
and chymostatin (final concentration: 5 ␮g mL⫺1, cell lysis
was performed by addition of Triton X-100 to a final concentration of 0.5%–1% [w/v]). After 5 min, efficiency of
lysis was checked microscopically and the particulate constituents removed by centrifugation for 15 min at 400,000g
in a Beckman (Munich, Germany) ultracentrifuge rotor
TLA 100.2. The supernatants were stored at ⫺75°C.
ODC Assay
ODC activity was determined by measuring the release
of 14CO2 from l-[1-14C]Orn (Schulz et al., 1985). No release
of 14CO2 from l-[1-14C]Orn was detected when the Chlamydomonas sp. lysates were pre-incubated in the presence of
the ODC inhibitor DFMO at a concentration of 0.1 mmol
L⫺1 for 10 min at 4°C before incubation with l-[1-14C]Orn.
One unit of ODC activity catalyzed the decarboxylation of
1 ␮mol of Orn min⫺1 at 37°C.
Determination of Protein
Quantitation of protein was performed by the method of
Minamide and Bamburg (1990) using bovine serum albumin as standard.
Isolation of Nuclei
Nuclei were prepared as recently described (Voigt and
Bohley, 2000) and stored at ⫺75°C as a suspension (1 ⫻ 109
nuclei mL⫺1) in a buffer containing 2.5% (w/v) Ficoll, 0.5
mol L⫺1 sorbitol, 20 mmol L⫺1 Tris-HCl (pH 7.5), 0.008%
(w/v) spermidine, 1 mmol L⫺1 dithiothreitol, 5 mmol L⫺1
MgCl2, and 50% (v/v) glycerol.
DNA Synthesis
DNA synthesis was measured in the absence and presence of aphidicolin (70 ␮mol L⫺1), an inhibitor of DNA
polymerase-␣, as previously described (Voigt and Münzner,
1987). The reaction mixture (20 ␮L) contained 55 mmol L⫺1
Tris-HCl (pH 8.0), 150 mmol L⫺1 KCl, 2.5 mmol L⫺1 MgCl2
0.002% (w/v) spermidine, 125 mmol L⫺1 sorbitol, 0.52%
(w/v) Ficoll, 12.5% (v/v) glycerol, 2.5 mmol L⫺1 dithiothreitol, 2.5% (v/v) dimethylsulfoxide, 1 mmol L⫺1 dCTP,
1 mmol L⫺1 dGTP, 1 mmol L⫺1 dTTP, 37 kBq [␣-32P] dATP
(110 Tbq/mmol), and 5 ⫻ 105 nuclei. After addition of
nuclei, the reaction mixture was incubated at 25°C for 1 h.
After incubation, 5 ␮L of stop mix, containing 5% (w/v)
SDS and 50 mmol L⫺1 EDTA, was added and the reaction
mixture plated onto 3-mm filters (Whatman Ltd., Maidstone, UK). The filters were washed once with 10% (w/v)
trichloroacetic acid (TCA), twice with 5% (w/v) TCA, and
twice with methanol, dried, and measured for radioactivity
after addition of 4 mL of UltimaGold (Packard Instruments, Groningen, The Netherlands). Blank values (reaction mixtures without incubation) were subtracted.
Plant Physiol. Vol. 128, 2002
Determination of Protein Biosynthesis
Biosynthesis of proteins was analyzed in vivo by pulse
labeling with [3H]Arg and measuring the radioactivity incorporated into protein. Aliquots (1 mL) were taken from
the cultures and incubated for 30 min in the presence of 370
kBq of [3H]Arg (specific radioactivity 1.7 Tbq mmol⫺1;
Amersham Pharmacia Biotech Europe GmbH, Freiburg,
Germany). After pulse labeling, the cells were rapidly
cooled to 0°C and centrifuged through a 0.4-mL cushion of
40% (w/v) Percoll in phosphate-buffered saline. The cells
were washed twice with 1 mL of phosphate-buffered saline
and finally dissolved in 100 ␮L of urea-SDS buffer containing 8 mol L⫺1 urea, 2% (w/v) SDS, 10 mmol L⫺1 EDTA, 200
mmol L⫺1 2-mercaptoethanol, and 20 mmol L⫺1 Tris-HCl,
pH 7.5. Aliquots (50 ␮L) were plated onto glass microfiber
filters (GF/C, Whatman Ltd.) and boiled for 15 min in 10%
(w/v) TCA to hydrolyze the aminoacyl-tRNAs. The filters
were then washed twice for 1 min with 5% (w/v) TCA,
twice for 1 min with 95% (v/v) ethanol, and finally with
diethylether. The dried filters were then measured for
radioactivity.
Analysis of Polyamines
Lyophilized samples were extracted in 5% (w/v) TCA
for 1 h in an ice bath and centrifuged for 30 min at 4°C and
20,000g in a Sorvall SS34 rotor. The supernatants were
evaporated at 70°C to 80°C. Dried samples were redissolved in 200 ␮L of 5% (v/v) perchloric acid and the
polyamines benzoylated according to Flores and Galston
(1982). Aliquots were analyzed by reversed-phase HPLC
according to Kotzabasis et al. (1993) using the HPLC system Gold (Beckman Instruments, San Ramon, CA)
equipped with an Ultraspere C18 column, 4.6 ⫻ 250 mm,
5-␮m particle size (Beckman, Meroue, Galway, UK). Elution of the benzoylpolyamines was performed at 25°C and
a flow rate of 1.0 mL min⫺1 with 60% (v/v) methanol and
monitored at 254 nm.
Received October 1, 2001; returned for revision November
13, 2001; accepted January 7, 2002.
LITERATURE CITED
Andersen SE, Bastola DR, Minocha SC (1998) Metabolism
of polyamines in transgenic cells of carrot expressing a
mouse ornithine decarboxylase cDNA. Plant Physiol 116:
299–307
Auvinen M, Paasinen A, Andersson LA, Hölttä E (1992)
Ornithine decarboxylase is critical for cell transformation. Nature 360: 355–358
Ben Hayyim G, Martin-Tanguy J, Tepfer D (1996) Changing root and shoot architecture with the rolA gene from
Agrobacterium rhizogenes: interactions with gibberillic
acid and polyamine metabolism. Physiol Plant 96:
237–243
Bercovich Z, Rosenberg-Hasson Y, Ciechanover A, Kahana C (1989) Degradation of ornithine decarboxylase in
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1477
Theiss et al.
reticulocyte lysate is ATP-dependent but ubiquitinindependent. J Biol Chem 264: 15949–15952
Bohley P (1996) Surface hydrophobicity and intracellular
degradation of proteins. Biol Chem 377: 425–435
Chattopadhyay MK, Ghosh B (1998) Molecular analysis of
polyamine synthesis in higher plants. Curr Sci 74:
517–522
Cvikrova M, Binarova P, Eder J, Vagner M, Hrubcova M,
Zon J, Machackova I (1999) Effect of inhibition of phenylalanine ammonia-lyase activity on growth of alfalfa
cell suspension culture: alterations in mitotic index, ethylene production, and contents of phenolics, cytokinins,
and polyamines. Physiol Plant 107: 329–337
Daoudi EH, Biondi S (1995) Metabolism and role of polyamines in plant development. Acta Bot Gall 142: 209–233
Davies DR, Plaskitt A (1971) Genetic and structural analyses of cell-wall formation in Chlamydomonas reinhardtii.
Genet Res Camb 17: 33–43
El Ghachtouli N, Martin-Tanguy P, Paynot M, Gianinazzi
S (1996) First report of the inhibition of arbuscular mycorrhital infection of Pisum sativum by specific and irreversible inhibition of polyamine biosynthesis or by gibberillic acid treatment. FEBS Lett 385: 189–192
Flores HE, Galston AW (1982) Polyamines and plant
stress: activation of putrescine biosynthesis by osmotic
shock. Science 217: 1259–1261
Fowler MR, Kirby MJ, Scott NW, Slater A, Elliot MC
(1996) Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in
suspension culture from quiescence to division. Physiol
Plant 98: 439–446
Fredlund JO, Johansson MC, Dahlberg E, Oredson SM
(1995) Ornithine decarboxylase and S-adenosylmethionine decarboxylase expression during the cell cycle of
chines hamster ovary cells. Exp Cell Res 216: 86–92
Galston AW, Kaur-Sawhney R, Altabella T, Tiburcio AF
(1997) Plant polyamines in reproductive activity and response to abiotic stress. Bot Acta 110: 197–207
Ghoda L, Sidney D, Macrae M, Coffino P (1992) Structural
elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation. Mol Cell Biol 12: 2178–2185
Graff JR, De Benedetti A, Olson JW, Tamez P, Casero RA,
Zimmer SG (1997) Translation of ODC mRNA and polyamine transport are suppressed in ras-transformed
CREFT cells by depleting translation initiation factor 4E.
Biochem Biophys Res Commun 240: 15–20
Harris EH (1989) The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego
Hayashi S, Murakami Y (1995) Rapid and regulated degradation of ornithine decarboxylase. Biochem J 306: 1–10
Hayashi S, Murakami Y, Matsufuji S (1996) Ornithine
decarboxylase antizyme: a novel type of regulatory protein. Trends Biochem Sci 21: 27–30
Hiatt AC, McIndoo J, Malmberg RL (1986) Regulation of
polyamine biosynthesis in tobacco: effects of inhibitors
and exogenous polyamines on arginine decarboxylase,
ornithine decarboxylase, and S-adenosylmethionine decarboxylase. J Biol Chem 261: 1293–1298
1478
Hibshoosh H, Johnson M, Weinstein IB (1991) Effects of
overexpression of ornithine decarboxylase (ODC) on
growth control and oncogene-induced cell transformation. Oncogene 6: 739–743
Jakus J, Wolff EC, Park MH, Folk EJ (1993) Features of the
spermidine-binding site of deoxyhyposine synthase as
derived from inhibition studies: effective inhibition by
bis- and mono-guanylated diamines and polyamines.
J Biol Chem 268: 13151–13159
Koza RA, Herbst EJ (1992) Deficiencies in DNA replication
and cell-cycle progression in polyamine-starved HeLa
cells. Biochem J 281: 87–93
Kotzabasis K, Christakis-Hampsas M, RoubelakisAngelakis JA (1993) A HPLC method for the identification and determination of free, conjugated and bound
polyamines. Anal Biochem 214: 484–489
Kotzabasis K, Senger H (1994) Free, conjugated and bound
polyamines during the cell cycle in synchronized cultures of Scenedesmus obliquus. Z Naturforsch 49: 181–189
Krupinska K, Humbeck K (1994) Light-induced synchronous cultures, an excellent tool to study the cell cycle of
unicellular algae. J Photochem Photobiol B: Biol 26:
217–231
Kumar A, Altabella T, Taylor MR, Tiburcio AF (1997)
Recent advances in polyamine research. Trends Plant Sci
2: 124–130
Li X, Coffino P (1992) Regulated degradation of ornithine
decarboxylase requires interaction with the polyamineinducible protein antizymes. Mol Cell Biol 12: 3556–3562
Li X, Coffino P (1993) Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamineinducible inhibitor protein. Mol Cell Biol 13: 2377–2383
Mamroud-Kidron E, Kahana C (1994) The 26S proteasome
degrades mouse and yeast ornithine decarboxylase in
yeast cells. FEBS Lett 356: 162–164
Manzella JM, Rychlik W, Rhoads RE, Hershey JWB,
Blackshear PJ (1991) Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure
and phosphorylation of eukaryotic initiation factors
eIF-4B and eIF-4E. J Biol Chem 266: 2383–2389
Marton LJ, Pegg AE (1995) Polyamines as target for therapeutic intervention. Annu Rev Pharmacol Toxicol 35:
55–91
Mihara S, Hase E (1971) Studies on the vegetative life cycle
of Chlamydomonas reinhardtii Dangeard in synchronous
culture: I. Some charasteristics of the cell cycle. Plant Cell
Physiol 12: 225–236
Minamide LS, Bamburg JR (1990) A filter paper dyebinding assay for quantitative determination of proteins
without interference from reducing agents and detergents. Anal Biochem 190: 66–70
Minocha SC, Minocha R (1995) Role of polyamines in
somatic embryogenesis. In YPS Bajaj, ed, Biotechnology
in Agriculture and Forestry, Vol 30: Somatic Embryogenesis and Synthetic Seed I. Springer-Verlag, Heidelberg,
pp 53–70
Moshier JS, Dosescu J, Skunca M, Luk GD (1993) Transformation of NIH/3T3 cells by ornithine decarboxylase.
Cancer Res 53: 2618–2622
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Polyamines Regulate Cell Division in Chlamydomonas reinhardtii
Murakami Y, Hayashi S (1985) Role of antizyme in degradation of ornithine decarboxylase in HTC cells. Biochem
J 226: 893–896
Pegg AE (1986) Recent advances in the biochemistry of
polyamines in eukaryotes. Biochem J 234: 249–262
Phillips MA, Coffino P, Wang CC (1987) Cloning and
sequencing of the ornithine decarboxylase gene from
Trypanosoma brucei. J Biol Chem 262: 8721–8727
Rechsteiner M, Rogers SW (1996) PEST sequences and regulation by proteolysis. Trends Biochem Sci 21: 267–271
Schlösser UG (1966) Enzymatisch gesteuerte freisetzung
von zoosporen bei Chlamydomonas reinhardtii Dangeard
in synchronkultur. Arch Microbiol 54: 129–159
Schulz WA, Gebhardt R, Mecke D (1985) Dexamethasone
restores hormonal inducibility of ornithine decarboxylase in primary cultures of rat hepatocytes. Eur J Biochem
146: 549–553
Slocum RD (1991) Tissue and subcellular localization of
polyamines and enzymes of polyamine metabolism. In
RD Slocum, HE Flores, eds, Biochemistry and Physiology
of Polyamines in Plants. CRC Press, Boca Raton, FL, pp
23–40
Surzycki S (1971) Synchronously grown cultures of
Chlamydomonas reinhardtii. Methods Enzymol 23: 67–73
Plant Physiol. Vol. 128, 2002
Tabor CW, Tabor H (1984) 1,4-Diaminobutane (putrescine), spermidine, and spermine. Annu Rev Biochem
53: 749–790
Voigt J, Bohley P (2000) Cell-cycle dependent regulation of
ornithine decarboxylase activity in the unicellular green
alga Chlamydomonas reinhardtii. Physiol Plant 110:
419–425
Voigt J, Deinert B, Bohley P (2000a) Subcellular localization and light-dark control of ornithine decarboxylase in
the unicellular green alga Chlamydomonas reinhardtii.
Physiol Plant 108: 353–360
Voigt J, Liebich I, Wöstemeyer J, Adam K-H, Marquardt
O (2000b) Nucleotide sequence, genomic organization
and cell-cycle-dependent expression of a Chlamydomonas
14–3-3 gene. Biochim Biophys Acta 1492: 395–405
Voigt J, Münzner P (1987) The Chlamydomonas cell cycle is
regulated by a light/dark-responsive cell-cycle switch.
Planta 172: 463–472
Voigt J, Münzner P (1989) An extranuclear gene contributes to the regulation of cell division of the unicellular
green alga Chlamydomonas reinhardtii. Plant Sci 59: 87–94
Walden R, Cordeiro A, Tiburcio AF (1997) Polyamines:
small molecules triggering pathways in plant growth
and development. Plant Physiol 113: 1009–1013
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1479