Plant Physiol. (1998) 116: 299-307
Metabolism of Polyamines in Transgenic Cells of Carrot Expressing
a Mouse Ornithine Decarboxylase cDNA1
Scott E. Andersen2,
Dhundy R. Bastola3, and
Subhash C. Minocha*
Department of Plant Biology, University of New Hampshire, Durham,
New Hampshire 03824
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ABSTRACT |
The
metabolisms of arginine (Arg), ornithine (Orn), and putrescine were
compared in a nontransgenic and a transgenic cell line of carrot
(Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [14C]Arg, [14C]Orn, and
[14C]putrescine were fed to cells and their rates of
decarboxylation, uptake, metabolism into polyamines, and incorporation
into acid-insoluble material were determined. Transgenic cells showed
higher decarboxylation rates for labeled Orn than the nontransgenic
cells. This was correlated positively with higher amounts of labeled
putrescine production from labeled Orn. With labeled Arg, both the
transgenic and the nontransgenic cells exhibited similar rates of
decarboxylation and conversion into labeled putrescine. When
[14C]putrescine was fed, higher rates of degradation were
observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn
decarboxylase pathway has no compensatory effects on the Arg
decarboxylase pathway, and (b) higher rates of putrescine production in
the transgenic cells are accompanied by higher rates of putrescine
conversion into spermidine and spermine as well as the catabolism of
putrescine.
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INTRODUCTION |
Polyamine metabolism has been implicated in regulating the level
and composition of the soluble nitrogen pools in plant cells (Tabor and
Tabor, 1984
; Smith, 1985; Altman, 1989; Evans and Malmberg, 1989; Bagni
and Pistocchi, 1990; Slocum, 1991). The three commonly occurring
polyamines (putrescine, spermidine, and spermine) are synthesized
largely from Orn and/or Arg. The rate-limiting step in the biosynthesis
of putrescine in animals and most fungi is the decarboxylation of Orn
by ODC (Fig. 1). In carrot (Daucus carota L.) cell cultures
and many other plants, putrescine is synthesized primarily via the
decarboxylation of Arg by ADC, followed by a series of intermediate
steps (Pegg, 1986; Minocha and Minocha, 1995; Kumar et al., 1997;
Walden et al., 1997). In some plants both ADC and ODC may be active,
with their activity being either tissue specific or developmentally
regulated. Putrescine is converted to spermidine with the addition of
an aminopropyl group derived from decarboxylated SAM through the
activity of spermidine synthase. The addition of another aminopropyl
group to spermidine by spermine synthase gives rise to spermine. In
most plants the cellular content of spermine is much lower than that of
spermidine or putrescine (Slocum, 1991; Minocha and Minocha, 1995).
Polyamines are catabolized mostly by diamine and polyamine oxidases
(Smith, 1985). Hormones, natural inhibitors, ozone, light, and
polyamines have all been shown to influence diamine and polyamine oxidase activities (Federico and Angelini, 1991). Polyamine oxidases are possibly also involved in the production of uncommon polyamines, e.g. norspermidine, norspermine, caldopentamine, caldohexamine, homocaldopentamine, and homocaldohexamine (Kuehn et al., 1990).
A variety of external and internal stimuli and the location of the
enzymes for putrescine biosynthesis may determine which pathway is
functional in different plant tissues at different developmental
stages. For example, it has been proposed that ODC may be active during
cell proliferation, and that ADC may be required for growth by
expansion and differentiation (Pandit and Ghosh, 1988). Also, it has
been shown that the ADC pathway is generally involved in putrescine
biosynthesis in response to stress
(Smith, 1990). Robie and Minocha
(1989) demonstrated that in the early stages of carrot somatic embryo
development, free putrescine was derived entirely via ADC, whereas ODC
was present only during the later stages of embryo development and
plant growth.
Previous studies of polyamine biosynthesis in relation to somatic
embryogenesis in our laboratory have focused on the role of auxin and a
potential competition between polyamine and ethylene biosynthesis for
SAM, which acts as a precursor for both pathways (see Minocha and
Minocha, 1995, and refs. therein). These studies have shown that (a) a
high cellular content of polyamines during somatic embryogenesis
correlates with the increased activities of ADC and SAMDC; (b) the ODC
activity increases only when mature, green somatic embryos are
produced; (c) the inhibition of ADC by
dl-
-difluoromethylarginine and of SAMDC by methylglyoxal
bis(guanylhydrazone) inhibits polyamine biosynthesis as well as somatic
embryogenesis; and (d) with the addition of
dl--difluoromethylornithine there is a promotion of ADC
and polyamines, an inhibition of ethylene, and a promotion in the
development of somatic embryos in the absence of 2,4-D. It has been
suggested that increased polyamine biosynthesis may promote somatic
embryogenesis via a reduction in ethylene production (Minocha and
Minocha, 1995).
More recently, transgenic carrot cell lines overexpressing a mouse ODC
cDNA have been produced (Bastola, 1994; Bastola and Minocha, 1995).
When the transgenic cells were placed on embryogenic medium, somatic
embryos formed earlier than in the control cells. This was correlated
with higher levels of putrescine in the transgenic cells (Bastola and
Minocha, 1995). The transgenic cells were able to grow and produce
somatic embryos in the presence of
dl--difluoromethylarginine (a strong inhibitor of ADC),
showing that the mouse ODC was providing sufficient quantities of
putrescine to the cells when the ADC pathway was inhibited. Exogenously
supplied polyamines actually inhibited somatic embryogenesis. It is
therefore believed that increased somatic embryogenesis is related to
the increased rates of polyamine biosynthesis and their fast turnover,
and not merely to the presence of high concentrations of polyamines in
the cells (Robie and Minocha, 1989; Minocha and Minocha, 1995). This
increased metabolism of polyamines, in turn, may affect the metabolism
of ethylene, ammonia, or both.
The present study was aimed at (a) a detailed examination of the
metabolism of Orn, Arg, and putrescine in nontransformed and in
transgenic cells of carrot expressing a mouse ODC cDNA, and (b)
determination of the compensatory effects of increased putrescine
production via ODC on the native ADC pathway. The results show that
increased production of putrescine via the mouse ODC in the transgenic
cells was accompanied by an increased catabolism of putrescine.
Furthermore, the ADC pathway was not significantly affected by the
increased production of putrescine via mouse ODC.
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MATERIALS AND METHODS |
Carrot Cell Cultures
Cell cultures of carrot (Daucus carota L.) were
maintained in B5 medium (Gamborg et al., 1968) supplemented with 2%
Suc and 2.3 µm 2,4-D. The medium was prepared from
premixed powder (catalog no. G-5893, Sigma) and adjusted to a pH of 5.5 before autoclaving. The medium was distributed to 500-mL Erlenmeyer
flasks at 200 mL per flask. Cultures were routinely transferred at 7-d
intervals. To maintain relatively small cell clumps, the cultures were
sieved every other week through a 250-µm sieve. Stock cultures were
also maintained on solid media (0.8% agar) that were transferred at 4-week intervals. Whereas the stock cultures of transgenic cells were
grown in media containing 300 mg/L kanamycin sulfate, the experimental
media did not contain kanamycin (Bastola and Minocha, 1995). The stock
cell suspensions were subcultured using a 10-mL-wide-bore Komagome
pipette (Iwaki Glass Co. Ltd, Tokyo, Japan), placing 10 mL of the
7-d-old cell suspension into 200 mL of the fresh B5 medium. All liquid
cultures were kept on a gyratory shaker at 155 rpm under 16 h of
fluorescent light (80 ± 10 µE m
2
s1) at 25 ± 2°C.
Experimental Setup
To prepare the experimental cultures, 7-d-old cell suspension was
centrifuged at 500g for 1 min in a 50-mL conical centrifuge tube. The cells were washed three times by centrifugation with fresh B5
medium without 2,4-D or kanamycin, and resuspended in 100 mL of B5
medium. After 3 d the cell suspensions were centrifuged at
500g for 1 min in 50-mL conical centrifuge tubes,
approximately one-half of the medium was discarded, and the cells were
resuspended in the remaining medium. Suspensions from several flasks
were pooled together in a round flask and dispensed with a Komagome pipette in 15-mL aliquots into 50-mL Erlenmeyer flasks. Each
experimental flask contained about 3.0 g of cells in 15 mL of
medium. To each flask, 100 µL of radioactive substrate (0.2 µCi)
was added. To measure decarboxylation, the flask was capped with a
rubber stopper holding a polypropylene well containing a 1.5- × 2-cm
piece of Whatman 3MM filter paper soaked with 50 µL of Scintigest
(Fisher Scientific) to adsorb
14CO2. The flasks were
incubated at 25°C and sampled at 1, 2, and 4 h for the
short-term experiments and at 4, 8, and 24 h for the long-term
experiments. The filter paper was removed from the well and counted for
radioactivity in 10 mL of Scintiverse (Fisher Scientific) for 10 min in
a liquid-scintillation counter (model LS 6000, Beckman). The
radioactive precursors used in this study were
[1,4-14C]putrescine (NEN; specific activity 117 mCi/mmol); l-[U-14C]Arg (Moravek,
Brea, CA; specific activity 270 mCi/mmol);
l-[U-14C]Orn (Amersham; specific
activity 257 mCi/mmol).
Collection of Cell Samples
At sampling times, 5-mL aliquots of the cell suspension were
removed and transferred to a vacuum-filtration unit fitted with Miracloth (Calbiochem) to collect the cells. The cells were washed once
with 1 mL of 5 mm unlabeled substrate and twice with 4 mL each of distilled H2O. For polyamine analysis,
1 g fresh weight of cells was added to 3 mL of ice-cold 5% PCA in
a chilled 16- × 100-mm disposable polypropylene tube. The cells were
frozen (
20°C) and thawed at room temperature three times to extract PCA-soluble metabolites (Minocha et al., 1994).
Dansylation of Polyamines for TLC
The procedure for dansylation and quantitation of polyamines was
modified from Minocha et al. (1994) to suit TLC separation using larger
quantities of tissue. The tubes containing frozen and thawed tissue in
PCA were centrifuged at 500g for 5 min. From the tubes, 1 mL
of PCA extract was removed and placed in a 5-mL glass Pyrex reaction
tube with a screw cap lined with a Teflon septum. A 1-mL sample of 0.4 mm standard polyamine mixture (putrescine, spermidine, and
spermine) was dansylated parallel with the samples. An equal amount of
saturated sodium carbonate was added to the reaction tube followed by 1 mL of dansyl chloride solution (20 mg/mL in acetone). The vials were
capped, vortexed for 15 s, and incubated in a 60°C water bath
for 1 h. The samples were then placed in a SpeedVac (Savant
Instruments, Farmingdale, NY) with the caps removed for 15 min to
evaporate acetone. Four-hundred microliters of Photrex-grade toluene
was added to each vial; the contents were mixed gently by inversion and
centrifuged for 1 min to separate the two phases. All of the toluene
phase was removed and placed in a microfuge tube. Aliquots of 50 µL
from both the toluene and the aqueous phase were counted separately in
10 mL of Scintiverse. The former count represented the total
dansyl-polyamines, whereas the latter count represented that which
either was not dansylated or was dansylated but charged at the high pH
so that it did not partition into the toluene fraction. This fraction would include the labeled amino acids and some of the polyamine metabolites.
Microfuge tubes containing the toluene fraction were placed in the
SpeedVac for 12 min to reduce the volume to about 50 µL. The
concentrated toluene fraction was spotted onto the adsorbent band at
the bottom of a Si250-PA silica gel TLC plate (J.T. Baker) and the
plate was air dried for 10 min. The plate was developed in a mixture of
cyclohexane:ethyl acetate (3:2, v/v). The solvent front ran for 45 min
to a distance of 3 cm from the top of the plate. The plate was removed
from the tank, allowed to dry for 5 min, and the dansyl-polyamine spots
were visualized under UV light. The spots were scraped with a
spatula, and the powder from each spot was mixed with 10 mL of
Scintiverse and counted for radioactivity.
Acid-Insoluble Fraction
The pellet from the PCA-extracted material was washed three times
with 5% PCA by centrifugation. One milliliter of 1 n NaOH was added to the tube, and the tube was incubated at 90°C for 1 h. From the tube, a 100-µL aliquot was counted for radioactivity. This fraction presumably contains the protein and cell wall-bound label
from the precursor and the metabolites.
Calculations, Statistical Analysis, and Data Presentation
After each incubation period, one-third of the radioactivity was
removed with the 5-mL aliquot of the cell suspension and a new filter
paper was placed in the well. Thus, the data for 14CO2 evolution experiments
are presented as a function of the time interval of incubation. On the
other hand, incorporation into the polyamines was calculated over the
entire length of the incubation period and the data are presented as
total dpm h1 g1 fresh
weight. Three replicate samples were collected at each time and each
experiment was repeated at least three times. The three repeat
experiments yielded similar results and data from a single
representative experiment are presented here. Since the endogenous
concentrations of metabolites were not measured in these experiments,
the data are presented directly as dpm and pertain only to the
metabolism of exogenously supplied radioactive substrate.
The data were analyzed using a Student's t-test in the
general linear model for one-way analysis of variance in Systat for Windows version 5.0 (SYSTAT Inc., Evanston, IL). In most cases, statistical comparisons were made between nontransgenic and transgenic cells only at a given time.
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RESULTS |
The transgenic cell line (ODC N14) used in this study was chosen
because of its high mouse ODC enzyme activity and high cellular putrescine content (Bastola and Minocha, 1995). In previous studies (Bastola and Minocha, 1995), the transgenic nature of this cell line
was established with respect to (a) presence of the mouse ODC gene, (b)
its expression at the transcriptional and translational levels, (c)
increased mouse-specific ODC activity, (d) increased putrescine
production, and (e) increased somatic embryogenesis on transfer to
2,4-D-free medium. These cells were maintained under the same
conditions as the nontransgenic cells except that kanamycin was added
to maintain selection pressure in the stock cultures of transgenic
cells, as stated in ``Materials and Methods''. Kanamycin was not present in the experimental media. The transgenic cells grew in the
presence of kanamycin at rates comparable to those in its absence, and
there were no significant effects of kanamycin on the cellular
polyamine content (data not presented here).
Polyamine Biosynthesis from Orn versus Arg
It is known that the only pathway for putrescine production in
carrot cells grown in culture is via ADC (Montague et al., 1978; Robie
and Minocha, 1989). These conclusions are based primarily on
measurements of ADC and ODC activity in cell extracts and not on the
basis of decarboxylation of labeled Arg or Orn or their incorporation
into polyamines by intact cells. In a series of experiments,
decarboxylation of [U-14C]Arg and
[U-14C]Orn were compared for the two cell lines
for a period of 24 h. Consistent with the results with studies
using cell extracts, the rates of decarboxylation of
[U-14C]Orn were found to be severalfold higher
in the transgenic cells as compared with nontransgenic cells (Fig.
2A). Significant differences were visible
within the 1st h of incubation and persisted through h 24 of
experimentation (data presented here only for the first 4-h period). On
the other hand, the rates of decarboxylation of [14C]Arg were not significantly different for
the two cell lines at any time except during the 1st h of incubation
(Fig. 2B).
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| Figure 2.
The rates of 14CO2
production from [U-14C]Orn (A) and
[U-14C]Arg (B) in nontransgenic (NT) and transgenic
(ODC-N14) carrot cells during a 4-h incubation period. Values are
means ± se of three replicates. Different lowercase
letters indicate that the values are significantly different (P  0.05) from each other at a given time. FW, Fresh weight.
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In both short-term and long-term experiments, the accumulation of
labeled putrescine derived from [U-14C]Orn was
severalfold higher in the transgenic cells than in the nontransgenic
cells (Fig. 3, A and B). Labeled
spermidine (Fig. 3, C and D) and spermine amounts (Fig. 3, E and F)
were also significantly higher in the transgenic cells as compared with
the nontransgenic cells. The amounts of label present in all three
polyamines increased with time in the transgenic cells but remained low
in the nontransgenic cells throughout the 24-h period. Among the three
polyamines, radiolabeled putrescine was the highest at any time
followed by the amounts of labeled spermidine and spermine in that
order.
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| Figure 3.
The incorporation of [U-14C]Orn into
labeled putrescine (A and B), spermidine (C and D), and spermine (E and
F) in nontransgenic (NT) and transgenic (ODC-N14) carrot cells during a
24-h incubation period. Dansylated polyamines were separated on TLC and
radioactivity was counted after elution of bands. The data for 0 to
4 h and 4 to 24 h were taken from two separate experiments.
Values are means ± se of three replicates. Different
lowercase letters indicate that the values are significantly different
(P 0.05) from each other at a given time. FW, Fresh weight.
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The amounts of labeled polyamines derived from
[U-14C]Arg were not significantly different
in the two cell lines at most of the times tested (Fig.
4). In the nontransgenic cells
radioactivity in all three polyamines increased with time up to 4 h. Thereafter, labeled putrescine levels showed a steady decline,
whereas the label in the other two polyamines remained high. In the
transgenic cells, on the other hand, labeled putrescine decreased with
time soon after the 1st h, whereas spermidine and spermine showed a steady accumulation of label in these fractions up to the 24 h.
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| Figure 4.
The incorporation of [U-14C]Arg into
labeled putrescine (A and B), spermidine (C and D), and spermine (E and
F) in nontransgenic (NT) and transgenic (ODC-N14) carrot cells during a
24-h incubation period. Dansylated polyamines were separated on TLC and
radioactivity was counted after elution of bands. The data for 0 to
4 h and 4 to 24 h were taken from two separate experiments.
Values are means ± se of three replicates. Different
lowercase letters indicate that the values are significantly different
(P 0.05) from each other at a given time. FW, Fresh weight.
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The total amount of labeled polyamines derived from
[U-14C]Orn was severalfold higher in the
transgenic cells as compared with the nontransformed cells at all times
tested (Table I). For
[U-14C]Arg, on the other hand, the contents of
labeled polyamines were quite comparable in the two cell lines. For
both [U-14C]Orn and
[U-14C]Arg, the highest amount of label was
generally present in putrescine. In the transgenic cells treated with
[U-14C]Orn, the amount of radioactivity in
spermidine was always less than 7% of that in total polyamines before
8 h. In contrast, almost 30% of the total label in polyamines was
present as spermidine in the nontransgenic cells. At 24 h of
treatment with [U-14C]Arg, labeled
spermidine was significantly greater in the transgenic cells as
compared to the nontransgenic cells. Moreover, the fraction of labeled
spermidine in the former was as high as 50% of the total label in
polyamines at 24 h of incubation. The relative proportion of
labeled spermine derived from [U-14C]Orn was
generally higher in the nontransgenic cells than the transgenic cells.
This, however, was not the case for spermine derived from
[U-14C]Arg.
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Table I.
Relative amounts of radiolabeled polyamines
collected up to 24 h in transgenic (N14) and nontransgenic (NT)
carrot cells when [U-14C]Orn or [U-14C]Arg
were used as precursors
Data presented here come from two separate experiments, one for 4 h and the other for 4 to 24 h.
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Uptake of Orn and Arg
The cellular pools of unused [14C]Orn and
[14C]Arg were estimated from the aqueous
fraction after dansylated polyamines had been extracted into toluene.
All polar metabolites and amino acids stay in this fraction while the
dansyl-polyamines partition into the toluene fraction. There was no
significant difference between the two cell lines in the amount of
labeled Orn present in the aqueous fraction at any time during the 4-h
incubation period (Fig. 5A). Moreover,
this fraction remained steady during the entire incubation period. The
amount of labeled Arg was comparable in the two cell lines and it also
remained unchanged during the 4-h incubation period (Fig. 5B).
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| Figure 5.
The amounts of label from [U-14C]
Orn (A) and [U-14C]Arg (B) present in the aqueous
fraction (remaining after the removal of dansyl-polyamines by toluene)
in nontransgenic (NT) and transgenic (ODC-N14) carrot cells during a
4-h incubation period. Values are means ± se of three
replicates. Different lowercase letters indicate that the values are
significantly different (P 0.05) from each other at a given
time. FW, Fresh weight.
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Incorporation of [14C]Orn and [14C]Arg
into Acid-Insoluble Material
To determine the amount of radioactivity that was being
incorporated into proteins or covalently bound to other macromolecules, the PCA-insoluble fraction of the cells was analyzed at the same time
as the soluble fraction. The transgenic cells had a significantly higher amount of both labeled Orn (Fig.
6A) and Arg (Fig. 6B) incorporated into
the acid-insoluble material in comparison with the nontransgenic cells
at all times. The amount of labeled Orn in the acid-insoluble material
increased with incubation time up to 4 h in the transgenic cells
(Fig. 6A) but remained unchanged throughout the experiment for the
nontransgenic cells. The incorporation of
[14C]Arg showed only a slight but statistically
insignificant change with the time of incubation in both cell lines
(Fig. 6B). It was generally higher in the transgenic cells than in
the nontransgenic cells. The incorporation of radioactivity into
acid-insoluble material for Arg was severalfold higher than that for
Orn at any time.
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| Figure 6.
The incorporation of [U-14C]Orn (A)
and [U-14C]Arg (B) into acid-insoluble material in
nontransgenic (NT) and transgenic (ODC-N14) carrots cells during a 4-h
incubation period. Values are means ± se of three
replicates. Different lowercase letters indicate that the values are
significantly different (P 0.05) from each other at a given
time. FW, Fresh weight.
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Metabolism of [1,4-14C]Putrescine
The experimental design for this part of the study was similar to
that employed for Arg and Orn metabolism. The rates of
14CO2 production, amount of
label present in the aqueous fraction, incorporation of label into
acid-insoluble material, and the conversion of putrescine into
spermidine and spermine were compared between the two cell lines over a
period of 24 h. The rates of
14CO2 produced from
[1,4-14C]putrescine in the transgenic cells
were significantly higher than those in the nontransgenic cells for the
periods of 0 to 4 h and 4 to 8 h (Fig.
7A). This difference, however, was not observed for the period of 8 to 24 h.
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| Figure 7.
The amount of 14CO2
released (A), 14C present in the acid-insoluble fraction
(B), and 14C present in the dansyl-polyamine fraction from
[1,4-14C]putrescine (C) in nontransgenic (NT) and
transgenic (ODC-N14) carrot cells during a 4-h incubation period.
Values are means ± se of three replicates. Different
lowercase letters indicate that the values are significantly different
(P 0.05) from each other at a given time. FW, Fresh weight.
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The amount of label appearing in the PCA-insoluble fraction that was
derived from [1,4-14C]putrescine was generally
similar in the two cell lines (Fig. 7B). The total amount of label in
this fraction did not change with time, indicating a continuous
turnover of this fraction. Figure 7C shows the amount of radioactivity
present in the total dansyl-polyamine fraction that partitioned into
toluene. The transgenic cells generally had significantly less label
than the nontransgenic cells at all times tested. The amount of label
in this fraction generally decreased with time.
As expected, putrescine was by far the most abundant of the three
polyamines at 4 h in both the cell lines followed by spermidine and spermine (Table II). Between 4 and
24 h, the relative proportion of labeled putrescine declined from
more than 70% in both cell lines to below 30%. By 24 h, the
proportion of label in spermidine in both cell lines was substantially
higher than that in putrescine. It increased from about 20% at 4 h to about 60% by 24 h. The amount of label in spermine also
increased with time but never exceeded 11% of the total label in
polyamines.
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Table II.
Relative amounts of radiolabeled polyamines
collected up to 24 h in transgenic (N14) and nontransgenic (NT)
carrot cells when [1,4-14C]putrescine was used as a
precursor
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DISCUSSION |
In the present study we compared the metabolism of putrescine and
its precursors Arg and Orn in a transgenic cell line constitutively expressing a mouse ODC cDNA and the control nontransgenic cell line. We
showed earlier that the transgenic cells contain significantly higher
concentrations of free putrescine with little change in free spermidine
and spermine (Bastola and Minocha, 1995). This was correlated with high
levels of mouse ODC-specific enzyme activity in the transgenic cells.
The results presented here provide a direct measurement of in vivo
enzyme activity of ADC and ODC in the transgenic as well as
nontransgenic cells. To our knowledge this type of information has not
been available before for these enzymes. As expected,
[14C]Orn was decarboxylated at a severalfold
higher rate in transgenic cells than in the nontransgenic cells. This
observation further shows that exogenously supplied Orn was rapidly
absorbed and became accessible as a substrate to the mouse ODC in
transgenic cells. The fact that the nontransgenic cells showed very
little decarboxylation of Orn is consistent with the low levels of
extractable ODC activity reported earlier for carrot cells (Montague et
al., 1978; Robie and Minocha, 1989; for review, see Minocha and
Minocha, 1995).
Except for the 1st h of incubation, the amount of
14CO2 produced from
[14C]Arg was similar in the two cell lines,
indicating that there was no general reduction in cellular ADC activity
in the transgenic cells. This is consistent with the ADC activity
measurements in transgenic carrot cells (Bastola and Minocha, 1995).
Similar results have been reported earlier by Hamill et al. (1990) in
Nicotiana rustica transgenic roots expressing a yeast ODC
gene, and by DeScenzo and Minocha (1993) in Nicotiana
tabacum cv Xanthi expressing the mouse ODC.
The relatively little change in putrescine biosynthesis via the ADC
pathway in cells that are producing large quantities of putrescine via
ODC is rather unexpected. It is commonly believed that there is a
strong homeostatic regulation of the polyamine biosynthetic pathway in
plants as well as animals, and this is achieved by feedback regulation
of the three decarboxylases, i.e. ADC, ODC, and SAMDC (for reviews, see
Pegg, 1986; Kumar et al., 1997; Walden et al., 1997). Data presented
here indicate no such feedback inhibition of ADC activity by putrescine
in carrot cells.
Based upon the amount of label in the aqueous fraction, it appears that
both labeled Orn as well as labeled Arg were taken up at similar rates
in the two cell lines. Moreover, these rates changed only slightly with
time. Thus, the observed differences in the rates of decarboxylation of
Orn and Arg, as well as their incorporation into putrescine in the two
cell lines, probably reflect the activities of the respective enzymes.
A potential problem with this argument, however, is that different
sizes of endogenous pools of these substrates would directly affect the amount of 14CO2 released at
short intervals. Cellular Orn and Arg pools in the transgenic cells
during normal growth conditions are not presently known. However, it is
conceivable that cellular Orn in the transgenic cells may be lower than
that in the nontransgenic cells because of its increased utilization by
mouse ODC (Bastola and Minocha, 1995). This could, therefore, result in
an overestimation of the rates of decarboxylation of
[14C]Orn as well as its conversion into
putrescine.
The metabolic fate of Orn involves (a) its conversion into putrescine
by ODC and subsequently into other polyamines, (b) its conversion into
citrulline and its derivatives, including Arg by Orn
carbamoyltransferase, and (c) its metabolism into
glutamate-
-semialdehyde and its derivatives, including Pro and Glu
by Orn transaminase (Slocum et al., 1984; Davis, 1986, and refs.
therein). Labeled Orn can be incorporated into acid-insoluble material
either as Arg or after catabolic conversion into Pro and Glu. A part of this fraction may include cell-wall-bound material. A large fraction of
Orn can also be stored unchanged in the vacuole from where it is only
slowly metabolized. The results presented here show that relatively
little of the radiolabeled Orn supplied from outside is converted into
Arg in the nontransgenic cells, since it does not appear as putrescine
in any significant amounts.
The data presented here provide further an insight into changes in the
metabolic flux of putrescine into spermidine and spermine. It has been
reported earlier by us and others that in plants (Hamill et al., 1990;
DeScenzo and Minocha, 1993; Bastola and Minocha, 1995) as well as in
animals (Halmekytö et al., 1995; Kauppinen and Alhonen, 1995;
Heljasvaara et al., 1997), increased putrescine biosynthesis by
transgenic overexpression of ODC has relatively little effect on the
cellular pools of spermidine and spermine. No studies have been
reported on the changes in metabolic turnover of these two polyamines
in the transgenic cells. Paulus and Davis (1981) found that whereas
[14C]Orn was quickly converted into putrescine
in Neurospora crassa, the latter remained sequestered for
several hours. Its conversion into spermidine and spermine and its
catabolism were relatively slow. It is also obvious from the published
literature that the cellular contents of spermidine and spermine in
plants are much more tightly regulated than those of putrescine (Hiatt
and Malmberg, 1988; Minocha et al., 1995; Minocha and Minocha, 1995).
Our results point to somewhat of a similar situation in that there was
only a slow appearance of labeled spermidine and spermine either from [14C]Orn or [14C]Arg.
The exogenously supplied [14C]putrescine was
also converted into spermidine and spermine at a slow rate. What most
of the previous studies do not reveal is the extent to which spermidine
and spermine turnover might be affected in relation to changes in
putrescine levels.
In spite of the overall low rates of conversion of putrescine into
spermidine and spermine, it is apparent from the data presented here
that the rates of spermidine and spermine biosynthesis were significantly higher in the transgenic cells as compared with the
nontransgenic cells when putrescine was produced from
[U-14C]Orn (Fig. 3) but not from
[U-14C]Arg (Fig. 4). If anything, the observed
amounts of labeled spermidine and spermine in the transgenic cells are
an underestimate of the actual rates of conversion of putrescine into
spermidine because of the fact that the pools of nonlabeled putrescine
in these cells are much higher (as much as 5- to 10-fold) than in the
nontransgenic cells (Bastola and Minocha, 1995). Since the amount of
radioactivity present in spermidine and spermine was always small
relative to the amount present in putrescine (whether synthesized from
[14C]Orn, [14C]Arg, or
provided exogenously as [14C]putrescine), it
can be argued that (a) the overall rates of biosynthesis of spermidine
and spermine in both cell lines were indeed low either due to the
limitation of decarboxylated SAM or the enzymes spermidine and/or
spermine synthase; or (b) newly synthesized spermidine was rapidly
turned over, so as not to affect its overall cellular levels. It is the
latter possibility that will cause an overall stimulation of polyamine
metabolism that may competitively affect the availability of SAM for
ethylene biosynthesis. Our preliminary investigation on the metabolism of [U-14C]Met does indeed show significantly
lower rates of its conversion into ethylene in the transgenic cells as
compared with the nontransgenic cells (S.E. Andersen and S.C. Minocha,
unpublished data).
At present, the fate of putrescine produced by ODC in transgenic cells
remains unclear. Mengoli et al. (1989) showed that exogenously supplied
putrescine was concentrated in the vacuole of carrot cells. It is not
known if the same happens to the mouse ODC-derived putrescine in the
transgenic cells. It is quite apparent that increased putrescine
production is correlated with its increased catabolism as measured by
14CO2 evolution from
[14C]putrescine as well as increased
incorporation of its label into PCA-insoluble material. The chemical
nature of the label from putrescine incorporated into the
acid-insoluble fraction is difficult to interpret because of the
binding of putrescine to the cell wall material, which is also present
in this fraction (Mengoli et al., 1989). In solanaceous plants,
putrescine is incorporated into many secondary compounds, such as
tropane alkaloids and pyrrolidine (Hamill et al., 1990; Altman and
Levin, 1993). Likewise, polyamines can be conjugated to various
phenolic and other secondary metabolites (Evans and Malmberg, 1989).
Although in some tissues the conjugated polyamines constitute a major
proportion of the total polyamines, carrot cell cultures have
polyamines mostly in the soluble form; only less than 10% are
conjugated (Minocha and Minocha, 1995). Rastogi and Davies (1989)
showed that in fruit pericarp discs of tomato, putrescine is
metabolized into spermidine, GABA, Glu, and a polar fraction eluting
with sugars and organic acids. Similarly, in Pinus radiata
cotyledons, Kumar and Thorpe (1989) showed the metabolism of putrescine
into GABA, aspartate, and glutamate; with GABA accounting for as much
as 24% of the total label. Using the dansylation procedure with
[14C]GABA, we found that GABA remained in the
aqueous fraction after partitioning with toluene (S.E. Andersen and
S.C. Minocha, unpublished data). In contrast to the possibility of
overestimation of decarboxylation of [14C]Orn
in the transgenic cells (due to low cellular Orn pools), the observed
catabolism of [14C]putrescine must be an
underestimation because of the fact that cellular pools of
nonradioactive putrescine are severalfold higher in the transgenic than
in the nontransgenic cells. Another possibility that is currently being
investigated in our laboratory to follow the fate of excessive
putrescine in transgenic cells is its secretion into the surrounding
medium. No information is currently available on the secretion of
polyamines by plant cells.
In conclusion, the results presented here show that (a) the higher
amount of putrescine in the transgenic cells is due to its increased
biosynthesis through the mouse ODC pathway; (b) the presence of
elevated cellular amounts of putrescine due to the mouse ODC has no
significant effect on the metabolism of Arg into putrescine by the
native ADC pathway, and (c) although cellular concentrations of
spermidine and spermine are not affected by the overproduction of
putrescine, their biosynthetic rates are, nevertheless, increased.
Furthermore, as suggested by Minocha and Minocha (1995), increased
putrescine production leads to an overall stimulation of the metabolic
pathway, which could adversely affect the biosynthesis of ethylene
through increased utilization of SAM. In this respect, studies are
underway to analyze the effects of increased polyamine metabolism on
the biosynthesis of ethylene in several transgenic cell lines of
carrot.
| |
FOOTNOTES |
1
This is scientific contribution no. 1959 from
the New Hampshire Agricultural Experiment Station.
2
Present address: Gene Discovery and Expression,
Monsanto, St. Louis, MO 63042.
3
Present address: Department of Biochemistry,
School of Medicine, University of Nevada, Reno, NV 89577.
*
Corresponding author; e-mail sminocha{at}christa.unh.edu; fax
1-603-862-3784.
Received May 2, 1997;
accepted September 18, 1997.
| |
ABBREVIATIONS |
Abbreviations:
ADC, Arg decarboxylase.
ODC, Orn
decarboxylase.
PCA, perchloric acid.
SAM, S-adenosylmethionine.
SAMDC, S-adenosylmethionine decarboxylase.
| |
ACKNOWLEDGMENTS |
The authors would like to thank Stephanie Long and Daniel
Coughlin for technical help and Dr. Curtis Givan, Dr. Rakesh Minocha, and Dr. Thomas Davis for helpful suggestions for improvement of the
manuscript.
| |
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Abstract
Introduction
