Journal of Experimental Botany, Vol. 57, No. 11, pp. 2589–2599, 2006
doi:10.1093/jxb/erl018 Advance Access publication 6 July, 2006
RESEARCH PAPER
Polyamine biosynthesis of apple callus under salt stress:
importance of the arginine decarboxylase pathway in
stress response
Ji-Hong Liu1,2, Kazuyoshi Nada3, Chikako Honda1, Hiroyasu Kitashiba1, Xiao-Peng Wen1,*,
Xiao-Ming Pang1 and Takaya Moriguchi1,†
1
National Institute of Fruit Tree Science, Tsukuba, Ibaraki, 305-8605 Japan
National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding,
Huazhong Agricultural University, Wuhan 430070, China
2
3
Faculty of Bioresources, Mie University, Tsu, Mie, 514-8507 Japan
Received 14 November 2005; Accepted 11 April 2006
Abstract
To clarify the involvement of the arginine decarboxylase (ADC) pathway in the salt stress response,
the polyamine titre, putrescine biosynthetic gene expression, and enzyme activities were investigated in
apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.)
Mansf.] in vitro callus under salt stress, during recovery after stress, and when ADC was inhibited by Darginine, an inhibitor of ADC. Salt stress (200 mM NaCl)
caused an increase in thiobarbituric acid-reactive substances (TBARS) and electrolyte leakage (EL) of the
callus, which was accompanied by an increase in free
putrescine content, during 7 d of treatment. Conjugated putrescine was also increased, but this increase
was limited to the early stage of salt stress. Accumulation of putrescine was in accordance with induction
of ADC activity and expression of the apple ADC gene
(MdADC). When callus that had been treated with 200
mM NaCl was transferred to fresh medium with (successive stress) or without (recovery) NaCl, TBARS and
EL were significantly reduced in the recovery treatment, indicating promotion of formation of new callus
cells, compared with the successive stress treatment.
Meanwhile, MdADC expression and ADC activity were
also decreased in the callus undergoing recovery,
whereas those of the callus under successive stress
were increased. Ornithine decarboxylase (ODC) activity
showed a pattern opposite to that of ADC in these
conditions. D-Arginine treatment led to more serious
growth impairment than no treatment under salt stress.
In addition, accumulation of putrescine, induction of
MdADC, and activation of ADC in D-arginine-treated
callus were not comparable with those of the untreated
callus. Exogenous addition of putrescine could alleviate salt stress in terms of fresh weight increase
and EL. All of these findings indicated that the ADC
pathway was tightly involved in the salt stress response. Accumulation of putrescine under salt stress,
the possible physiological role of putrescine in alleviating stress damage, and involvement of MdADC and
ADC in response to salt stress are discussed.
Key words: Arginine decarboxylase (ADC), electrolyte leakage,
Malus sylvestris var. domestica, MdADC, putrescine, salt
stress, thiobarbituric acid-reactive substances.
Introduction
Plants encounter adverse environmental stresses during
their life cycle, which have negative impacts on growth
and greatly affect crop productivity. As perennial crops,
fruit trees are exposed to an array of stresses for a long time
once they are planted. If the trees are severely injured by
* Present address: Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang 550025, China.
y
To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: ADC, arginine decarboxylase; AIH, agmatine iminohydrolase, CPA, N-carbamoylputrescine amidohydrolase; CPM, callus proliferation
medium; EL, electrolyte leakage; MDA, malonyldialdehyde; ODC, ornithine decarboxylase; PCA, perchloric acid; TBA, thiobarbituric acid; TBARS,
thiobarbituric acid-reactive substances; TCA, trichloroacetic acid.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
2590 Liu et al.
environmental stresses, it would be hard for them to
recuperate, leading to retarded growth and reduced fruit
production. Furthermore, the negative effects of the stresses
are not limited to the fruit production in the current year but
can also extend to the next year(s). Therefore, it is of critical
importance to develop techniques for reducing stress injury
and/or improving stress tolerance in fruit trees for sustainable cropping, which can be fulfilled by the cultivation
method or genetic engineering (Holmberg and Bülow,
1998). In both cases, elucidation of the mechanisms of the
stress responses of fruit trees is needed. However, little information is available on fruit trees compared with annual
plants due to, in part, recalcitrant manipulations possibly
caused by a high degree of heterozygosity and a long
juvenile period.
In order to survive and grow, plants attempt to cope with
the stresses by initiating some defensive machinery that has
been gradually unravelled in model plants (Chinnusamy
et al., 2005). Recently, changes in the polyamines,
spermidine and spermine, together with their diamine
precursor, putrescine, have been suggested as an important
response of plants under a variety of environmental stresses
(Bouchereau et al., 1999). Putrescine is generally known to
increase under many stress conditions, whereas spermidine
and spermine frequently remain unchanged or show minor
changes (Bouchereau et al., 1999). In higher plants,
putrescine is biosynthesized directly from decarboxylation of ornithine through ornithine decarboxylase (ODC,
EC 4.1.1.17) or indirectly from arginine through arginine
decarboxylase (ADC, EC 4.1.1.19). Apart from the accumulation of putrescine, abiotic stresses have been shown to
increase ADC activity and induce the accumulation of ADC
gene expression (Borrell et al., 1996; Perez-Amador et al.,
2002; Hummel et al., 2004). Moreoever, impairment of
ADC led to more serious cell injury, as has been revealed in
the mutant of the AtADC2 gene (adc2-1) in Arabidopsis
created by Ds insertion (Urano et al., 2004). These pieces
of evidence suggest that the ADC pathway may play a key
role in stress response. However, it is noted that such
a contention arose predominantly from annual crops,
whereas relevant information is very limited in perennial
crops despite the existence of a few reports so far (Kushad
and Yelenosky, 1987; Tang and Newton, 2005). Since
elucidation of the stress response is equally as important for
stable growth and development of perennial crops, it is
necessary to identify how their ADC pathway functions
under abiotic stress.
Salt stress is one of the most devastating abiotic stresses,
which influences 20% of the world’s cultivated land and
nearly all of the irrigated lands, and seriously interrupts
normal plant growth and productivity (Flowers, 2004).
Consequently, elucidation of the stress response under
a salt environment has prompted attention worldwide. In
a previous work, the apple ADC gene, MdADC, was isolated and it was shown that its expression was up-regulated
by salt stress (Hao et al., 2005). However, it remains to be
investigated how the ADC pathway, in terms of ADC
activity, MdADC expression, and its product (putrescine), is
involved in the salt stress response of apple, a representative
of perennial crops. In addition, it is also unclear how this
pathway functions in the case of stress removal and ADC
inhibition. Therefore, to obtain a deeper insight, using apple
callus, the aim of the present study was to investigate: (i)
the effects of salt stress on growth of apple callus during
7 d of stress; (ii) the effects of stress removal on growth
of apple callus following 7 d of stress; and (iii) the effects
of D-arginine, an inhibitor of ADC, and putrescine on the
response of apple callus to salt stress. Callus cultures were
employed herein because they proliferate rapidly and are
easy to handle. The results indicated that salt stress
impaired callus growth and increased cell injury, coupled
with a conspicuous increase in putrescine content through
the induction of MdADC expression and ADC activity,
because no ODC expression was detected and ODC activity
did not parallel the stress damage. Inhibition of the ADC
pathway by D-arginine further exacerbated the callus injury,
which could be alleviated by exogenous putrescine, implying an intimate relationship between the ADC pathway and
stress response in apple callus. It was shown that the ADC
pathway may be a prevalent pathway in both perennial and
annual crops in response to salt stress. To our knowledge,
this is the first report on the comprehensive elucidation of
the ADC pathway under salt stress in perennial crops,
which will be of interest for generating stress-tolerant
germplasms through genetic manipulation.
Materials and methods
Plant material
In vitro callus of apple [Malus sylvestris (L.) Mill. var. domestica
(Borkh.) Mansf. cv. Orin] induced from young fruits was maintained
at 2-week intervals on callus proliferation medium (CPM) containing
MS salt (Murashige and Skoog, 1962), NN organic component (Nitsch
and Nitsch, 1969), 3% sucrose, 4.5 lM 2,4-dichlorophenoxyacetic
acid, 1 lM N6-benzylaminopurine, and 0.8% agar (pH 5.8) in the
dark at 25 8C. The callus did not show morphogenesis in the present
culture system.
Treatments of in vitro callus
After subculture for four cycles, 2-week-old callus was used for three
different experiments. In experiment I, callus was cultured on CPM
supplemented with 200 mM sodium chloride (NaCl) for 7 d, while
the callus cultured on CPM alone was considered as the control.
The callus was sampled at day 0, 6 h, and at days 1, 3, and 7. In
experiment II, callus that had been subjected to 200 mM NaCl for 7 d
was transferred to fresh CPM with (successive stress) or without
(recovery) 200 mM NaCl, and the callus was harvested 6 h and 1 d
after the transfer. In experiment III, callus was cultured for 7 d on
CPM, CPM+200 mM NaCl, CPM+5 mM D-arginine, CPM+5 mM
D-arginine+200 mM NaCl, and CPM+5 mM D-arginine+200 mM
NaCl+5 mM putrescine. In another set of experiments, callus was
cultured on CPM+200 mM NaCl and CPM+200 mM NaCl+
putrescine (1 or 5 mM) for 7 d. After autoclaving the CPM media,
Polyamine biosynthesis of apple callus under salt stress
with or without the addition of NaCl, filter-sterilized stocks of
D-arginine and Put, separately or in combination, were added as
needed. In experiment III, callus was sampled on the last day of the
experiment. Fresh weights of the callus were measured at the onset
and completion of the experiment in experiments I and III.
Thiobarbituric acid-reactive substances (TBARS) and electrolyte
leakage (EL) were measured at each sampling time in experiments
I and II, while only EL was measured in experiment III. The sampled
callus was frozen in liquid nitrogen and stored at ÿ80 8C until used
for the extraction of polyamines, enzymes, and RNA.
Measurement of TBARS and EL
Lipid peroxidation of the callus was determined as the amount of
MDA (malonyldialdehyde) measured by the thiobarbituric acid
(TBA) reaction as reported by Tang et al. (2004) with minor
modifications. About 0.1 g of callus was homogenized in 1 ml of
20% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged for 20 min at 5000 g (room temperature). To 1 ml of the
supernatant, 1 ml of 20% TCA containing 0.5% TBA (w/v) and
100 ll of 4% butylated hydroxyl toluene in ethanol (v/v) were added.
The mixture was heated at 95 8C in a water bath for 30 min and
then cooled down rapidly on ice for 20 min. After centrifugation for
5 min at 10 000 g, the absorbance of the supernatant (MDA–TBA
complex) was measured at 532 nm by a UV-visible spectrophotometer (UV-1600, Shimadzu, Japan) and the value for non-specific
absorption at 600 nm was corrected. The concentration of MDA was
calculated as described before (Heath and Packer, 1968).
EL measurement was performed based on the method of Liu
et al. (2004). A callus of 0.5 g was incubated in 20 ml of distilled
water and kept at room temperature for 1 h, and the initial conductivity
(C1) was measured by a conductivity meter (Horiba, DS-52,
Japan). The samples were then boiled for 10 min to induce
100% leakage, followed by cooling at room temperature for 12 h
and measurement of final electrolyte conductivity (C2). The relative
conductivity (C) was expressed as a percentage of the total ion leakage and calculated as C (%)=1003C1/C2.
Quantification of polyamines by HPLC
Free polyamines were quantified according to a previous method
with minor modifications (Liu et al., 2006). To extract polyamines,
;0.1 g of callus was homogenized in 1 ml of 5% perchloric acid
(PCA) and extracted on ice for 30 min. After centrifugation for 5 min
at 5000 g (4 8C), the supernatant was transferred to another tube
and kept on ice. The pellet was extracted again with 1 ml of 5% PCA
on ice for 30 min and then centrifuged under the same conditions.
The supernatant from the two centrifugations was combined, from
which 100 ll were transferred to a 0.5 ml tube and adjusted to pH
7.0 with saturated Na2CO3. An 80 ll aliquot of the neutralized
extract was then transferred to a glass V-vial, followed by addition
of 10 ll of 1 M sodium borate buffer (pH 6.3) and 100 ll of 5 mM
9-fluorenylmethyl chloroformate in acetone. After vortexing for 1 min
at room temperature, 200 ll of 1-adamantanamine in acetone
was added and vortexed again for another 30 s. The mixture was
then centrifuged for 5 min at 5000 g (4 8C). A 10 ll aliquot of
the supernatant was subjected to high-performance liquid chromatography (HPLC; Prominence Series, Shimadzu, Japan) for free
polyamine quantification. The pellet of the extracted sample was
washed with 5% PCA three times and then suspended in 250 ll of
5% PCA. For measurement of the conjugated and bound polyamines,
an aliquot of 250 ll from the combined supernatant and the suspended
pellet (250 ll) was hydrolysed separately in 6 M HCl for 18 h at
110 8C. The hydrolysate was then dried at 70 8C with a flow of
nitrogen. The resulting residues were dissolved in 500 ll of 5%
PCA, followed by measurement with the same method as for free
polyamines.
2591
RNA isolation and RNA gel blotting
Total RNA (10 lg) isolated from callus according to the procedure
described by Wan and Wilkins (1994) was electrophoresed in a
1.2% formaldehyde denatured agarose gel. RNA was blotted onto
Hybond N membrane (Amersham Bioscience, NJ, USA) and hybridized with digoxigenin-labelled partial ADC (pMdADC) probes
according to the method described by Hao et al. (2005). Hybridization was carried out at 42 8C in high SDS buffer containing 7% SDS,
0.05 M phosphate (pH 7.0), 53 SSC, 1% blocking reagent, and 0.1%
N-lauroylsarcosine. After hybridization, the membranes were washed
twice for 5 min each with 23 SSC+0.1% SDS at room temperature,
and then twice for 15 min each with 0.53 SSC+0.1% SDS at 65 8C.
Addition of antibody, membrane washing, equilibration, and treatment by chemiluminescent substrate were performed according to the
manual provided by the manufacturer (Roche Diagnostics, Germany).
The relative value of the expression level was corrected to the rRNA
level by densitometric analysis (Scion Image, Scion Corporation,
MD, USA). Since no signals of ODC were detected in apple callus
regardless of salt treatment (data not shown), only the expression of
MdADC is presented herein.
Analysis of enzyme activities
Polyamine biosynthetic enzymes, ADC and ODC, were extracted
from 1–5 g samples in extraction solution composed of 100 mM
potassium phosphate (pH 8.0) containing 20 mM sodium ascorbate,
1 mM pyridoxal-59-phosphate, 10 mM dithiothreitol, 0.1 mM EDTA,
and 0.1 mM phenylmethylsulphonyl fluoride. The homogenate was
centrifuged at 25 000 g for 20 min. Enzyme activity assay was carried
out in 3.5 ml Pierce vials sealed with snap caps according to a
previous report (Song et al., 2001). Three 5 mm filter paper discs,
saturated with 2 N KOH, were fixed onto a syringe needle that was
inserted through the cap into the vial, in which a reaction mixture
containing 100 ll of enzyme extracts plus 10 ll of 5 mM pyridoxal59-phosphate with 1.85 kBq of L-[1-14C]arginine (2.03 TBq molÿ1,
American Radiolabeled Chemicals Inc., MO, USA), 1.85 kBq of L[1-14C]ornithine (2.03 TBq molÿ1, American Radiolabeled Chemicals Inc.), for ADC and ODC, respectively, was present. After
incubation for 60 min at 30 8C, 0.2 ml of 10% TCA was added to stop
the reaction, which was incubated for an additional 90 min. The filter
paper discs were then removed, and, after drying, were put into liquid
scintillation cocktail and counted for radioactivity using a liquid
scintillation counter (Aloka LSC-5100, Japan). Enzyme activity was
quantified according to Bradford (1976) using bovine serum albumin
as a standard.
Statistical analysis
All of the treatments were done at least in duplicate with three
repetitions in each, and data typical of the trends are presented. The
data were mean values of three repetitions, expressed as the mean 6
SE. The statistical analysis was performed by one-way analysis
of variance (ANOVA), taking P <0.05 as significant.
Results
Experiment I: effects of salt stress on growth of
apple callus
‘Orin’ callus induced from young fruits was light yellowish
and granulated. When cultured on CPM, it showed good
proliferation capacity and increased its fresh weight steadily
over a 14 d culture, in which the fastest growth occurred in
the middle stage of the culture (data not shown). When the
2592 Liu et al.
callus was treated with 200 mM NaCl for 7 d, its growth
was severely retarded since the fresh weight increase of the
salt-treated callus was ;6-fold lower than that of the
control (data not shown). In addition, salt-stressed callus
became somewhat brown compared with the control, which
implied that salt treatment has caused severe impairment
of growth of in vitro callus.
Since TBARS and EL were considered as good indicators of stress-derived cell injury, they were assayed in the
control and salt-treated callus. TBARS of the control callus
showed minor change during a 1 week culture, ;30 nmol
gÿ1 FW (Fig. 1A). In contrast, salt caused an increase in
TBARS at 6 h, reaching nearly 40 nmol gÿ1 FW at day 1,
which increased continuously at day 3 (47 nmol gÿ1 FW),
and day 7 (53 nmol gÿ1 FW), significantly (P <0.01) higher
than that of the control. Similarly, EL of the control did
not show any notable change during the whole process,
whereas salt led to a sharp rise 6 h after the treatment. EL
of the salt-treated callus continued to rise at day 1, followed
by a slow increase at days 3 and 7; but it was still
significantly higher (P <0.01) than that of the control
(Fig. 1B). At any given sampling time, TBARS and EL in
the salt-treated callus were higher than those in the control.
Free and conjugated polyamines were the most abundant
types in the callus, whereas bound polyamines were
negligible (data not shown). Regardless of the polyamine
type, putrescine was the most predominant fraction,
followed by spermidine and spermine. In order to know if
the polyamine levels of callus shown herein truly reflected
endogenous levels, free polyamines in the medium sampled
from the control and salt treatment were checked. Although
three fractions could be detected in the medium, their levels
were quite low in comparison with those in the callus,
suggesting that the polyamines detected in the samples
were the same as endogenous levels. Changes in free
putrescine of the control and the salt-treated callus exhibited a similar trend, i.e. an increase from day 0 to day 3,
followed by a negligible decrease at day 7 (Fig. 2A).
However, at any given time point, the salt-treated callus
showed higher putrescine levels than the control. For
instance, putrescine in the stressed callus was 3053 and
5709 nmol gÿ1 FW at days 1 and 3, respectively,
significantly higher (P <0.05) than those of the control
(1091 nmol gÿ1 FW at day 1 and 3284 nmol gÿ1 FW at day
3). After transfer to fresh CPM medium, apple callus
showed the accumulation of a large amount of conjugated
putrescine, from 3734.5 nmol gÿ1 FW at 6 h to 11 277 nmol
Fig. 1. Changes in thiobarbituric acid-reactive substances (A) and
electrolyte leakage (B) in the apple callus during 7 d of culture on
CPM with (NaCl) or without (control) 200 mM NaCl. Data were the
mean 6SE (n=3). * Significantly different at P <0.05; ** significantly
different at P <0.01 based on one-way ANOVA.
Fig. 2. Changes in free and conjugated putrescine (A), free spermidine,
and free spermine (B) in the apple callus during 7 d of culture on CPM
with (NaCl, filled bars) or without (control, open bars) 200 mM NaCl.
Data were the mean 6SE (n=3). * Significantly different at P <0.05 based
on one-way ANOVA.
Polyamine biosynthesis of apple callus under salt stress
2593
ÿ1
g FW at day 7. Conjugated putrescine levels in the salt
treatment were much higher than those in the control at 6 h
and at day 1 (P <0.05); this decreased sharply at day 3,
followed by a rise to the same level as the control at day 7
(Fig. 2A), implying that conspicuous accumulation of
conjugated putrescine was revealed only at the early stages.
Both free spermidine and free spermine were at very low
levels compared with free putrescine. No difference in free
spermidine was observed between the control and the salttreated callus (Fig. 2B). Spermine of the control showed no
variations during the course of the experiments, while that
of the salt-treated callus was significantly increased from
day 3 onwards (Fig. 2B). Conjugated spermidine was also
at low levels, while conjugated spermine was not detected
with the analytical method used here (data not shown).
The change in total polyamines (free plus conjugated) in the
salt-treated callus showed a pattern similar to that of
the conjugated putrescine (data not shown).
The steady-state level of MdADC mRNA in the control
and the stressed callus was monitored from day 0 to day
7 (Fig. 3A). The callus without salt stress (control) showed
no obvious change in MdADC gene expression from day
0 to day 3, and increased at day 7. When the callus was
exposed to 200 mM NaCl, the level of MdADC transcript was sharply increased 6 h after the stress treatment,
which was maintained until day 7.
ADC activity of the control was not detected until
day 3, followed by an abrupt increase at day 7 (Fig. 3B).
When the callus was treated with 200 mM NaCl, ADC activity could be detected at 6 h, and increased to the peak
value at day 3, followed by a decrease at day 7. The activity
of salt-treated callus was significantly higher than its
control counterpart before the last sampling time. ODC
activity showed the same trend in both the control and salttreated callus; it decreased until day 1 and increased up to
day 7 (Fig. 3C). Unlike ADC, however, the activity of ODC
in the control was significantly higher than that in the salttreated callus during the procedure, except at day 1.
Experiment II: effects of stress removal on
apple callus response
In experiment I, it was demonstrated that salt stress led
to a rise in TBARS, EL, MdADC expression, and ADC
activity as early as 6 h after salt stress, suggesting a rapid
response following the treatment. Therefore, attempts were
made to determine if these factors responded rapidly, i.e. at
6 h and 1 d after stress relief. It is not easy to track changes
in callus proliferation during a short period. As an alternative, TBARS and EL of callus transferred to fresh
CPM with (successive stress) or without (recovery) 200
mM NaCl after 7 d of salt stress were used as indicators of
cell injury so as to identify if removal of salt stress could
affect the lipid peroxidation and cell injury. TBARS were
reduced slightly 6 h after the callus was transferred to the
Fig. 3. Expression of MdADC (A), ADC activity (B), and ODC activity
(C) in the apple callus during 7 d of culture on CPM with (NaCl) or
without (control) 200 mM NaCl. Ethidium bromide staining showed
equal loading of rRNA, and the relative values of the expression level
normalized to the rRNA level by densitometric analysis are shown in (A).
The hybridization signal at day 0 was set to 100 and the other signals were
quantified accordingly. Enzyme activities were the mean 6SE (n=3).
* Significantly different at P <0.05; ** significantly different at
P <0.01 based on one-way ANOVA.
recovery medium, which was maintained 1 d after the
transfer, whereas TBARS of the successively stressed
callus was significantly higher (P <0.05) than the recovery
counterparts 6 h and 1 d after transfer (Fig. 4A). Similarly,
removal of salt led to a remarkable decrease in EL at both
sampling times, and was significantly lower (P <0.01) than
that of the successively stressed callus (Fig. 4B), indicating
that removal of salt stress might have led to the recovery
of a lot of the cells from salt-derived injury and thereby
promoted formation of new callus cells possibly through
the activation of cell division.
MdADC expression was also compared between the
recovery and the successively stressed callus (Fig. 5A).
2594 Liu et al.
Fig. 4. Changes in thiobarbituric acid-reactive substances (A) and
electrolyte leakage (B) 6 h and 1 d after transfer of the apple callus that
has been stressed with 200 mM NaCl for 7 d (NaCl 7d) to fresh medium
with (successive stress) or without (recovery) salt. Data were the mean
6SE (n=3). * Significantly different at P <0.05; ** significantly different
at P <0.01 based on one-way ANOVA.
Culture of the callus on recovery medium for 6 h caused
a reduction in MdADC transcription level, which was
maintained at day 1, indicating that the response of MdADC
to salt stress was very quick. However, successive stress
led to an increased expression level 6 h and 1 d following
the transfer relative to the initial stage and its recovery
counterpart. Signal normalization demonstrated that the
callus of recovery and successive stress showed nearly
60% and 120% relative mRNA intensity of the initial
material, respectively.
In the recovery callus, ADC activity was not affected 6 h
after the transfer, but it was decreased by 22.2% at day 1
(Fig. 5B). However, transfer of the 7 d salt-stressed callus
to medium for successive stress caused an increase in
ADC activity at 6 h (16.7%) and day 1 (38.9%), which was
significantly higher than the activity in the corresponding
recovery callus (P <0.05). As far as ODC activity was
Fig. 5. Expression of MdADC (A), ADC activity (B), and ODC activity
(C) 6 h and 1 d after transfer of the apple callus that has been stressed
with 200 mM NaCl for 7 d (NaCl 7d) to fresh medium with (successive
stress) or without (recovery) salt. Ethidium bromide staining showed
equal loading of rRNA, and the relative values of the expression level
normalized to the rRNA level by densitometric analysis are shown in (A).
The hybridization signal of the apple callus stressed by 200 mM NaCl for
7 d (NaCl 7d) was set to 100 and the other signals were quantified
accordingly. Data were the mean 6SE (n=3). * Significantly different at
P <0.05; ** significantly different at P <0.01 based on one-way ANOVA.
concerned, it showed a pattern different from that of ADC,
since activities of the recovery callus were significantly
higher (P <0.01) than those in the successively stressed
callus 6 h and 1 d after the transfer (Fig. 5C). However, no
difference in polyamine titres was observed between the
recovery callus and the successively stressed callus at both
sampling times (data not shown).
Experiment III: effects of D-arginine and putrescine on
the response of apple callus to salt stress
As the growth and cell injury were paralleled by MdADC
expression and ADC activity rather than ODC, D-arginine,
an ADC inhibitor, was used to investigate how salt stress
affected polyamine biosynthesis of apple callus with or
without exogenous putrescine. When callus was cultured
Control, CPM medium; NaCl, CPM+200 mM NaCl; D-Arginine, CPM+5 mM D-arginine; D-Arginine+NaCl, CPM+5 mM D-arginine+200 mM NaCl; D-Arginine+NaCl+Put, CPM+5 mM
mM NaCl+5 mM putrescine. After autoclaving the CPM media, with or without the addition of NaCl, filter-sterilized stocks of D-arginine and Put, separately or in combination,
were added as needed. Data, derived from a representative experiment, are means 6SE (n=3). ND, not detected.
a
D-arginine+200
1.660.1
12.160.1
8.060.3
13.660.7
7.564.0
58.360.3
69.462.0
68.462.9
95.367.1
96.5634.9
3623.96 197.8
5097.06773.5
1549.96121.2
1686.7622.7
5857.26681.1
0.3960.02
0.1560.04
0.4260.04
0.5860.07
0.3260.13
0.7260.02
0.5760.02
ND
ND
ND
49.669.6
92.961.3
56.765.8
90.860.3
77.067.3
121.565.5
18.361.9
32.765.5
11.966.7
31.763.2
Control
NaCl
D-Arginine
D-Arginine+NaCl
D-Arginine+NaCl+Put
Putrescine
Spermidine
Spermine
12 762.362263.7
11954.062047.0
7401.0651.8
6923.16772.8
10 061.261447.1
Conjugated putrescine
(nmol gÿ1 FW)
Free polyamines (nmol gÿ1 FW)
ODC activity
(pmol 14CO2 mgÿ1
protein sÿ1)
ADC activity
(pmol 14CO2 mgÿ1
protein sÿ1)
EL (%)
FW increase (%)
Treatmentsa
Table 1. Fresh weight (FW) increase, electrolyte leakage (EL), ADC and ODC activities, and polyamine contents of apple callus cultured on different media for 7 d
Polyamine biosynthesis of apple callus under salt stress
2595
on CPM supplemented with D-arginine, its growth in terms
of fresh weight increase was obviously reduced, as indicated by only 32.7% growth compared with 121.5% in
the control (Table 1). Salt treatment of the D-argininetreated callus caused a more serious reduction in the growth
(11.9%), which was returned to the level of D-argininetreated callus by exogenous putrescine at 5 mM (31.7%).
No difference in EL was detected between the control and
D-arginine-treated callus, although it was slightly higher in
the latter. Salt stress imposed on D-arginine-treated callus
led to a sharp increase in EL (90.8%) relative to the
D-arginine-treated callus, which was reduced to a lower
value (77.0%) by exogenous putrescine. Addition of 5 mM
D-arginine to CPM for 7 d completely inhibited ADC
activity. Application of salt stress alone or both salt and
exogenous putrescine did not induce ADC activity in the
D-arginine-treated callus. However, ODC activity could
be detected in the D-arginine-treated callus, and it was
slightly higher than in the control. ODC activities in the
D-arginine-treated callus subjected to either salt or salt and
putrescine in combination were not affected appreciably.
D-Arginine treatment of apple callus caused a reduction
in free and conjugated putrescine, but increased free
spermidine and spermine to different extents (Table 1).
Conjugated spermidine did not change significantly, and
conjugated spermine was not detected (data not shown).
When the D-arginine-treated callus was exposed to salt
stress, both free and conjugated putrescine showed a minor
change, which was enhanced notably by addition of
exogenous putrescine.
D-Arginine treatment led to a remarkable reduction in the
transcription level of MdADC (Fig. 6, D5) compared with
the untreated control (N0). The expression of MdADC in
the D-arginine-treated callus (D5) was negligibly induced
upon salt stress (D5 N200), whereas callus without Darginine treatment under salt stress (N200) showed much
stronger expression. Exogenous addition of putrescine to
callus with the combined treatment of D-arginine and salt
increased the expression level (D5 N200 P5). This showed
that MdADC expression paralleled the changes in free and
conjugated putrescine.
These results demonstrated that inhibition of ADC by
D-arginine led to more serious damage under salt stress
and that exogenous putrescine could improve the growth
of D-arginine-treated callus in response to salt stress.
However, the alleviation effects of exogenous putrescine
were possibly due to its reversal of the damage caused by
D-arginine inhibition rather than by antagonizing the stress
directly. Thus, a further investigation was carried out to
determine if the exogenous addition of putrescine could
truly alleviate salt stress in the absence of D-arginine, as
reported elsewhere (Tang and Newton, 2005). As shown in
Table 2, the exogenous addition of putrescine at 1 and
5 mM to the stress medium (CPM+200 mM NaCl) led to
a 33.1% and 34.9% increase in fresh weight, respectively,
2596 Liu et al.
Fig. 6. Expression of MdADC in apple callus that has been cultured for 7
d on CPM (control, N0), CPM+5 mM D-arginine+200 mM NaCl (D5
N200), CPM+5 mM D-arginine (D5), CPM+200 mM NaCl (N200), and
CPM+5 mM D-arginine+200 mM NaCl+5 mM putrescine (D5 N200 P5).
Ethidium bromide staining showed equal loading of rRNA, and the
relative values of the expression level normalized to the rRNA level by
densitometric analysis are shown. The hybridization signal of the control
was set to 100 and the other signals were quantified accordingly.
Table 2. Effects of exogenous putrescine on growth and
electrolyte leakage of apple callus
Data, derived from a representative experiment, are the mean6SE (n=3).
Different letters in the same column indicate significant difference based
on one-way ANOVA (P <0.05).
Treatmentsa
Fresh weight increase (%)
Electrolyte leakage (%)
NaCl
NaCl+Put 1
NaCl+Put 5
19.366.4 a
33.165.3 b
34.962.8 b
93.360.7 a
86.265.4 b
85.265.2 b
NaCl, CPM+200 mM NaCl; NaCl+Put 1, CPM+200 mM NaCl+1 mM
putrescine; CPM+Put 5, CPM+200 mM NaCl+5 mM putrescine. All of
the media also contained 0.8% agar and 3% sucrose. After autoclaving
the CPM media with NaCl, the filter-sterilized stock of Put was added
as needed.
values significantly higher (P <0.05) than found for the
stress medium (19.3%). Exogenous putrescine at both
concentrations also significantly reduced EL of the stressed
callus. Both a decrease of EL and the coincident increase
in growth of salt-treated callus by exogenous putrescine
demonstrated its capacity to alleviate stress-derived
damage.
Discussion
During the growth of apple callus cultured on CPM without
salt treatment, high free putrescine titres were detected (Fig.
2A), which may be relevant to meeting the requirements for
cell division since polyamines have been proposed to be
correlated with this physiological process (reviewed by
Slocum et al., 1984). In addition, a high level of conjugated
putrescine was also observed during normal growth of the
callus, ;2–7 times that of its free counterpart. The relative
proportions of free and conjugated polyamines varied from
species to species, and detection of a high level of
conjugated polyamines has been reported in tobacco
(Slocum and Galston, 1985; Torrigiani et al., 1987) and
Prunus avium (Garin et al., 1995). For instance, the amount
of conjugated putrescine of P. avium cotyledons cultured in
vitro was 16 801 nmol gÿ1 FW, almost 200 times that of the
free putrescine (Garin et al., 1995). Although conjugated
polyamines have been detected in a wide range of plants,
their physiological significance is still ambiguous. It
has previously been proposed that conjugation may be a
way of regulating cellular free polyamines and the conjugated
polyamines serve as a reserve of the free forms (reviewed
by Martin-Tanguy, 1985). Nevertheless, a concurrent increase in both free and conjugated putrescine in the control
callus reported here did not underpin such a hypothesis.
It remains elusive whether the conjugated putrescine is
also implicated in cell division, in a similar manner to
free putrescine, as has been proposed elsewhere (Bonneau
et al., 1994).
A large number of reports have demonstrated accumulation of polyamines under a variety of environmental
stresses, such as salt, drought, extreme temperature, acidity,
ozone, and heavy metals (reviewed by Bouchereau et al.,
1999). In the present study, marked accumulation of free
putrescine was observed in apple callus under stress, which
is in accordance with the case for tobacco cell lines
(Kuthanová et al., 2004), oat leaf (Tiburcio et al., 1994),
Arabidopsis seedlings (Urano et al., 2003), and pepino
fruits (Martı́nez-Romero et al., 2003). The increase in free
polyamines might be due to de novo synthesis through the
induction of MdADC expression and ADC activity in apple
callus, as discussed below. As well as free putrescine,
accumulation of conjugated putrescine was also observed
in salt-treated callus at an early stage, followed by a decline
at days 3 and 7 (Fig. 2A). Compared with the intensive
attention which has been focused on changes in free
polyamines in response to stress, alteration of the conjugated form has been less well characterized although stressinduced accumulation of conjugated polyamines has been
reported previously (Martı́nez-Romero et al., 2003; Liu
et al., 2004, 2005; Camacho-Cristóbal et al., 2005). The
mechanism underlying the accumulation of large levels
of conjugated putrescine at an early stage of salt stress
may either be a stress response or cell defence, as has been
delineated by Bors et al. (1989). However, a rapid decline
of conjugated putrescine at days 3 and 7 in the salt-treated
callus was not anticipated. If the total (free plus conjugated)
polyamines are considered as a global potential, the abrupt
decline might possibly be related to homeostatic equilibrium of cellular polyamines so that the cells did not
accumulate polyamines to a level causing cell toxicity,
indicating that the cells could evolve some undetermined
mechanisms, for example, activation of the 4-amino-nbutyric-acid (GABA) pathway through oxidation (Bagni
and Tassoni, 2001), to regulate internal polyamines.
It should be noted that, in addition to the accumulation
of free putrescine, a decrease of free putrescine in response
to stresses has also been observed in some species, depending
on the stress type, duration, and plant species (Santa-Cruz
et al., 1997). It needs to be investigated why different polyamine patterns occur under stresses. One possible
Polyamine biosynthesis of apple callus under salt stress
reason is the disparate sensitivity/tolerance to stresses of the
plant species. Krishnamurthy and Bhagwat (1989) reported
that rice cultivars sensitive to salt accumulated putrescine
under salt stress, whereas tolerant cultivars accumulated
predominantly spermidine and spermine. Herein, accumulation of putrescine, along with negligible changes in
spermidine and spermine, in the apple callus was in line
with the report on rice (Krishnamurthy and Bhagwat,
1989), indicating that apple callus in the present work
might be classified as a sensitive type. Although it still
remains ambiguous whether accumulation of putrescine
under stress is a general response in sensitive species,
several lines of evidence have shown that spermidine or
spermine is more effective for alleviating stress-induced
damage than putrescine (Velikova et al., 1998). In addition,
the accumulation of cellular putrescine might be a consequence of stress rather than a protective mechanism, as has
been suggested by Botella et al. (2000). Therefore,
putrescine that accumulated in the cells of sensitive species,
without concordant accumulation in spermidine and spermine, might have few if any profound effects on counteracting the stress, which may explain why the callus grew
slowly and showed serious loss of fresh weight despite
accumulation of a large quantity of putrescine under salt
stress in experiment I. Nevertheless, the callus suffered
damage in a more severe manner under salt stress when the
endogenous putrescine was reduced by D-arginine (experiment III), implying the presence of a putrescine threshold
contributing to normal cellular functions.
The present results demonstrated that exogenous putrescine could alleviate salt stress-induced damage. Fresh
weight increases of the callus cultured on stress medium
supplemented with putrescine were higher than those
without putrescine, together with decreased EL (experiment
III). It is evident that exogenous putrescine supplied to Darginine-treated callus increased the endogenous form as
well. Therefore, the alleviation could be attributed to the
increase in putrescine derived from uptake of exogenous
putrescine since spermidine showed no change and spermine was reduced (Table 1). It is generally accepted that
polyamines including putrescine are highly protonated at
a physiological pH, which favours electrostatic binding of
polyamines to negatively charged components of membranes, leading to membrane stabilization through ionic
interactions (Slocum et al., 1984). In addition, putrescine
may be effective in scavenging free radicals, as has been
shown elsewhere (Drolet et al., 1986). Recently, several
reports suggested that exogenous polyamines could activate
the antioxidant systems thereby controlling free radical
generation and preventing membrane lipid peroxidation,
which resulted in improved cell growth under stress (Tang
and Newton, 2005; Verma and Mishra, 2005), in agreement with the reduction in EL reported here. Although the
exact mode of action of putrescine could not be identified
in the present study, it was clearly shown that exogenous
2597
putrescine alleviated the stress damage. As perennial plants,
fruit trees are vulnerable to a diverse set of environmental
stresses such as salt, drought, and chilling. The present data
showed that exogenous application of polyamines can
alleviate stress-derived damage and may find practical use
in protecting seedlings or scions against stress, as has been
reported by Tang and Newton (2005) who successfully
demonstrated that exogenous polyamines reduced saltinduced oxidative damage to Virginia pine plantlets, leading to improved growth and development of plantlets in the
presence of salt. However, a question raised is why the
internal putrescine induced upon stress (experiment I) did
not have the same effects on alleviating the stress injury as
the exogenous putrescine. The possibility exists that different compartmentation of the putrescine took place in
these situations. Since ADC has been shown to be mainly
localized in the chloroplasts and nuclei at the cellular
level (Borrell et al., 1995; Bortolotti et al., 2004), internal
putrescine biosynthesis might occur primarily in these
organelles. Upon stress without exogenous putrescine, the
accumulated part may be limited to where it is produced
with little transported to the membrane, the site of injury.
However, exogenous putrescine may be taken up by the
cells through the membrane, where it can bind sufficiently
to the negatively charged functional groups and, consequently, protect them against the stress (Slocum et al.,
1984).
Our previous work has demonstrated that growth of
apple callus depended predominantly on putrescine biosynthesized through the ADC but not the ODC pathway
(Hao et al., 2005). In this study, a decrease in free putrescine of apple callus treated with D-arginine was concomitant with a loss of ADC activity and an increase in ODC
activity (experiment III), further supporting the proposal.
Moreover, ODC expression could not be detected in apple
callus and ODC activity did not parallel stress parameters
such as callus growth, TBARS, and EL, which compelled
the hypothesis that ODC also contributed little to stress
response. The discrepancy between ODC gene expression
and ODC activity might be due to the presence of a different
type of ODC gene with low homology to the known ODC
genes in other plant species, as reported previously (Hao
et al., 2005). An increase in the putrescine titre of apple
callus under salt stress was accompanied by a marked
increase in MdADC expression and ADC activity, which,
however, rapidly decreased when apple callus was transferred to medium without salt (experiment II). On the other
hand, when ADC was inhibited by D-arginine, salt stress
caused no activation of ADC activity, negligible induction
of MdADC, and little change in putrescine, along with more
serious growth inhibition (11.9% versus 18.3%). Such a
phenomenon is in accordance with the work on Arabidopsis thaliana mutants (spe1-1 and spe2-1) with reduced
ADC activity, where salt stress caused more severe loss in
fresh weight, coupled with less putrescine accumulation, of
2598 Liu et al.
the mutants compared with the wild type (Kasinathan and
Wingler, 2004). ADC has been referred to as a general
stress enzyme since its activity is induced by a myriad of
stresses (Nam et al., 1997; Legocka and Kluk, 2005). In
addition, the gene coding for ADC has been shown to be
a key element under a variety of stresses (Soyka and Heyer,
1999; Urano et al., 2004). Perez-Amador et al. (2002) also
reported that ADC2 was the only gene of polyamine biosynthesis involved in the wounding response. All
of these findings, taken together, seemed to indicate that
ADC and/or the MdADC gene was responsive to the stress
milieu and important for putrescine formation in apple
callus under stress. Nevertheless, it has to be pointed
out that changes in ADC activity and MdADC transcript
were not completely consistent with putrescine titres, as is
shown in Figs 2A and 3A where strong activation of ADC
activity in the salt-treated callus between days 3 and
7 was not accompanied by corresponding increases in
free putrescine. Similarly, the MdADC level remained constant 6 h after salt stress (Fig. 3A), whereas free putrescine increased progressively until the end of salt treatment
(Fig. 2A). Lack of consistency under these circumstances may be attributable to either regulation of polyamines at different levels or complex regulation processes
such as synthesis, degradation, and oxidation (Palavan
and Galston, 1982).
It is worth mentioning that in the ADC pathway another
two enzymes, agmatine iminohydrolase (AIH, also known as
agmatine deiminase, EC 3.5.3.12) and N-carbamoylputrescine
amidohydrolase (CPA, EC 3.5.1.53), are also involved in
putrescine biosynthesis. Genes encoding these two enzymes have been cloned in A. thaliana (Janowitz et al.,
2003; Piotrowski et al., 2003). To date, no information is
available concerning expression of the AIH gene upon
stress, but it was shown that the transcription level of the
CPA gene was not altered by osmotic stress that caused
strong induction of ADC2, indicating that CPA might not
be the rate-limiting enzyme in stress-derived putrescine
accumulation (Piotrowski et al., 2003). Since putrescine
synthesis is mediated indirectly by ADC, overexpression of
the ADC gene may lead to an increased putrescine pool
(Roy and Wu, 2001). If the accumulated putrescine can be
further pushed forward to cause an increase in spermidine
and spermine, stress tolerance will be anticipated, as has
been demonstrated by improved drought tolerance in
transgenic rice overexpressing the heterologous Datura
stramonium ADC gene (Capell et al., 2004). This will
open up new avenues for enhancing crop tolerance to
abiotic stresses by modulating polyamine biosynthesis
through transgenic manipulation of the ADC gene.
Acknowledgement
This work was supported in part by a Grant-in Aid from the Japan
Society for Promotion of Science (JSPS).
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