Plant Cell Physiol. 49(4): 617–624 (2008)
doi:10.1093/pcp/pcn033, available online at www.pcp.oxfordjournals.org
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The Signaling Role of Extracellular ATP and its Dependence on Ca2þ Flux
in Elicitation of Salvia miltiorrhiza Hairy Root Cultures
Shu-Jing Wu, Yuan-Shuai Liu and Jian-Yong Wu *
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, PR
China
important roles in several physiological processes. In animal
cells, ATP is well established as an extracellular signal in a
number of cellular responses, such as neurotransmission,
immune response, apoptosis and the regulation of blood
pressure (Zheng et al. 1991, Bours et al. 2006). The
extracellular ATP (eATP) signal may be transmitted
across the plasma membrane into the interior of the
cell via specific nucleotide receptors or purinoceptors. The
signal processes induced by eATP in animal cells include
the production of reactive oxygen species (ROS) and nitric
oxide (NO), and an increase in the intracellular Ca2þ
concentration (Shen et al. 2005, Bours et al. 2006).
The signaling role of eATP in plant cells was brought
up very recently and has so far been examined in only a few
studies (Demidchik et al. 2003, Jeter et al. 2004, Song et al.
2006). Nevertheless, the experimental results provide strong
support for the signaling role of eATP in plant cell stress
responses. One of the most important findings was the rapid
and transient increase in the cytosolic Ca2þ concentration
upon the application of exogenous ATP to Arabidopsis root
or seedlings (Demidchik et al. 2003). The increase in
cytosolic Ca2þ concentration due to plasma membrane
influx or release from intracellular stores is an early event in
plant response to biotic or abiotic elicitors, and Ca2þ acts as
a second messenger in the elicitor signal transduction
(Blumwald et al. 1998, Zhao et al. 2005). The eATP also
induced several other important events in stress signaling
and response, such as the production of ROS, and the
activation of mitogen-activated protein kinases (MAPKs),
lipoxygenase (LOX, a key enzyme for jasmonic acid
biosynthesis) and ACS6 (a key enzyme for ethylene
biosynthesis) (Jeter et al. 2004, Song et al. 2006).
Experimental evidence is also available for the ATP release
into or the presence in the extracellular matrix (ECM) of
intact plant tissues in Arabidopsis cell cultures (Chivasa
et al. 2005) and Medicago truncatula root hairs (Kim et al.
2006), and the stimulated ATP release by touch and osmotic
stress in Arabidopsis seedlings (Jeter et al. 2004). It has been
suggested that the ATP release from plant cells may be
accomplished by passive means resulting from plasma
The application of a polysaccharide elicitor from yeast
extract, YE, to Salvia miltiorrhiza hairy root cultures induced
transient release of ATP from the roots to the medium,
leading to a dose-dependent increase in the extracellular ATP
(eATP) level. The eATP level rose to a peak (about 6.5 nM
with 100 mg l1 YE) at about 10 h after YE treatment, but
dropped to the control level 6 h later. The elicitor-induced
ATP release was dependent on membrane Ca21 influx, and
abolished by the Ca21 chelator EGTA or the channel blocker
La31. The YE-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane
purinoceptors. On the other hand, the application of
exogenous ATP at 10–100 kM to the cultures also induced
rapid and dose-dependent increases in H2O2 production and
medium pH, both of which were effectively blocked by RB and
EGTA. The non-hydrolyzable ATP analog ATPcS was as
effective as ATP, but the hydrolyzed derivatives ADP or
AMP were not so effective in inducing the pH and H2O2
increases. Our results suggest that ATP release is an early
event and that eATP plays a signaling role in the elicitation of
plant cell responses; Ca21 is required for activation of the
elicitor-induced ATP release and the eATP signal transduction. This is the first report on ATP release induced by a
fungal elicitor and its involvement in the elicitor-induced
responses in plant cells.
Keywords: Ca2þ flux — Extracellular ATP — Fungal
elicitor — Hþ flux — H2O2 production — Salvia
miltiorrhiza.
Abbreviations: ABC, ATP-binding cassette; eATP, extracellular ATP; ECM, extracellular matrix; MS, Murashige–Skoog;
PPADS, pyridoxalphosphate-6-azophenyl-20 , 40 -disulfonic acid;
RB, reactive blue; ROS, reavtive oxygen species; YE, polysaccharide elicitor from yeast extract.
Introduction
ATP is the universal energy source for biochemical
reactions in all living organisms, and also plays other
*Corresponding author: E-mail, [email protected]; Fax, þ852-23649932.
617
Extracellular ATP signaling in elicitor response
Results
YE-induced ATP release and dependence on Ca2þ flux
After the addition of YE into the hairy root cultures, a
notable increase of the ATP concentration in the culture
medium (eATP) was detected within 30 min. The eATP
concentration rose to a peak in about 10 h, and then
dropped back to the initial control level 6 h later (Fig. 1A).
The eATP level increased with the YE dose, and the peak
level at 100 mg l1 was about 6.5 nM, which was 70 times
higher than the control level. The YE-induced ATP release
was completely blocked by both EGTA (5 mM) and La3þ
(2 mM) (Fig. 1B). The results show that YE induced a
transient release of ATP from the hairy roots in a dosedependent manner, and the induction of ATP release by YE
was dependent on plasma membrane Ca2þ influx.
A
8
ATP (nM)
membrane disruption, and by active means such as the
ATP-binding cassette (ABC) proteins through the anion
channels, and the cell exocytosis of ATP-containing vesicles
and subsequent vesicular fusion (Thomas et al. 2000,
Kim et al. 2006).
However, the findings regarding the signaling role of
eATP from previous studies have been mainly based on the
events evoked by exogenous ATP applied to the plant
tissues instead of the eATP released from plant cells in
response to external stimuli. There is still no reported study
on the ATP release induced by a microbial elicitor and on
the involvement of eATP in the elicitor-induced responses.
Plant defense response signal transduction is mediated by
multiple signaling pathways involving numerous signal
molecules (Zhao et al. 2005). Although numerous signaling
agents in plants have been documented, the role of eATP as
a signaling agent is of special and more general interest as
ATP is the ubiquitous energy source in all living organisms
and is abundant in plant cells. In addition to stress
responses, eATP may play a signaling role in diverse
physiological processes in plant cells, like its roles in
animal cells.
The aim of this study was to examine the signaling role
of eATP in elicitor-induced responses in the hairy root
culture of Salvia miltiorrhiza Bunge (Lamiaceae). Salvia
miltiorrhiza root is a valuable Chinese herb (Danshen)
which is widely used for the treatment of menstrual
disorders and cardiovascular diseases (Tang and
Eisenbrand 1992, Wang et al. 2007). Hairy root culture of
S. miltiorrhiza has been established as a potential alternative
to farm growth of whole plants for efficient production of
Danshen and its bioactive components, the diterpenoid
tanshinones (Hu and Alfermann 1993, Shi et al. 2007). The
polysaccharide fraction of yeast extract (YE) was used as an
elicitor in this study to induce the responses of the hairy
roots in liquid culture.
Control
YE10mg/l
YE50
YE100
6
4
2
0
B 8
Control
YE100
YE + La2
YE+EGTA5
6
ATP (nM)
618
4
2
0
0
4
8
12
16
20
Time (h)
Fig. 1 Elicitor YE-induced ATP release in S. miltiorrhiza hairy root
cultures (A); the inhibition of YE-induced ATP release by Ca2þ
antagonists (B) (EGTA at 5 mM and LaCl3 at 2 mM, added to
the culture 0.5 h before YE addition). Error bars indicate the SD
at n ¼ 3.
YE- and eATP-induced H2O2 production and dependence
on Ca2þ flux
YE induced rapid production of H2O2 in the hairy root
cultures, leading to an increase in the H2O2 concentration in
the medium. The production of H2O2 was detectable within
10 min after YE addition to the culture, and reached the
maximum level in about 4 h (Fig. 2A). The YE-induced
H2O2 production was effectively (nearly completely)
blocked by the eATP inhibitor RB (100 mM) and completely
blocked by the Ca2þ chelator EGTA (10 mM) (Fig. 2A),
suggesting the strong dependence of YE elicitation of H2O2
on both the eATP signal and the Ca2þ influx across the
plasma membrane. The rapid and transient production of
ROS such as H2O2 known as the oxidative burst is an early
event in plant cell response to biotic and abiotic elicitors or
stresses, and H2O2 also plays an important role (as a second
messenger) in the elicitor signal transduction.
The supply of exogenous ATP to the hairy root culture
also induced rapid H2O2 production, detectable within
10 min of the eATP supply (Fig. 2B). The H2O2 concentration showed a small increase between 0 and 0.5 h after the
eATP supply, a period of slow and gradual increase between
0.5 and 3 h, and another notable increase to a peak at 4 h.
Extracellular ATP signaling in elicitor response
5
H2O2 (µM)
4
Control
YE100mg/L
YE+EGTA10
YE+RB40
0.2
Change of medium pH
A
YE+RB100
3
2
1
0.1
Control
YE100mg/L
YE+EGTA10
YE+RB20
EGTA10
YE+RB50
0
−0.1
−0.2
−0.3
0
0
1
2
3
4
5
6
Time (h)
B
3
Control
ATP10 µM
ATP40 µM
ATP100 µM
ATP40+RB50
ATP40+EGTA5
Fig. 3 Elicitor YE-induced pH change (drop) in the hairy root
culture medium and the effects of EGTA (10 mM) and RB (20 and
50 mM).
2
1
0
0
1
2
3
Time (h)
4
5
6
Fig. 2 H2O2 production induced by YE (A) and exogenous ATP
(B), and the inhibition by the Ca2þ chelator EGTA and the eATP
inhibitor RB in the hairy root cultures. EGTA and RB were added to
the culture 0.5 h before YE; EGTA number is the dose in mM, RB
number is the dose in mM.
The eATP-induced H2O2 production in the hairy root
cultures was completely inhibited by RB (100 mM) and
EGTA (10 mM) (Fig. 2B), suggesting its dependence on the
membrane nucleotide receptors (blocked by RB) and the
Ca2þ membrane influx (blocked by EGTA).
YE- and eATP-induced pH change and dependence on
Ca2þ flux
The elicitor YE caused a rapid and notable drop in the
medium pH, by up to 0.25–0.3 units at 6 h after its addition
to the hairy root cultures, and the YE-induced pH drop
could not be blocked by either the Ca2þ chelator EGTA or
the eATP inhibitor RB (Fig. 3). In contrast, the supply of
eATP to the hairy root cultures caused a rapid increase in
the medium pH, detectable within 10 min of the eATP being
supplied (Fig. 4A). The ATP-induced pH increase was most
rapid and significant at 100 mM, reaching a maximum of
0.27 pH units in 30 min (Fig. 4A). The eATP-induced pH
increase was completely blocked by both EGTA at 10 mM
(Fig. 4A) and RB at 100 mM, and was partially blocked by
RB at lower doses of 20 and 50 mM (Fig. 4B). The results
suggest the dependence of the eATP-induced pH increase
Change of medium pH
A 0.4
0.3
Control
ATP20
ATP40
ATP100
ATP100+EGTA5
0.2
0.1
0
B
Change of medium pH
H2O2 (µM)
619
0.3
0.2
Control
ATP+RB20µM
ATP+RB50
RB50
ATP+RB100
0.1
0
0
1
2
3
Time (h)
4
5
6
Fig. 4 Exogenous ATP-induced medium pH increase and the
inhibition by EGTA and RB in the hairy root cultures. ATP and RB
numbers are the doses in mM, and EGTA number is the dose in mM.
on plasma membrane Ca2þ influx and the membrane
nucleotide receptors. The inhibitors EGTA and RB alone
also caused slight changes in the medium pH by no more
than 0.03 units, which was negligible compared with that
caused by YE or eATP.
Effects of ATP derivatives, PPADS and apyrase on medium
pH and H2O2 production
An additional control experiment was conducted with
the non-hydrolyzable form of ATP, ATPgS, and two
620
Extracellular ATP signaling in elicitor response
5
A 0.4
ATP
Control
YE100 mg/L
ATPγS
ADP
YE+Apyase10
YE+Apyase25
AMP
4
YE+PPADS
ATP+PPADS
H2O2 (µM)
pH change
0.3
Control
0.2
0.1
0
H2O2 (µM)
3
0
1
2
3
Time (h)
4
5
6
Fig. 6 Effects of PPADS and apyrase on YE-induced H2O2
production. Apyrase was supplied at 10 or 25 U ml1 and PPADS
at 100 mM; PPDAS or apyrase alone induced no significant
responses.
2
1
0
2
1
0
B
3
0
1
2
3
4
5
6
Time (h)
Fig. 5 Medium pH change (A) and H2O2 production (B) induced
by the ATP analog and derivatives, and the inhibition of ATPinduced responses by PPADS. All reagents were supplied at
100 mM to the hairy root cultures; PPDAS alone induced no
significant responses.
hydrolyzed ATP derivatives, ADP and AMP, for the above
ATP-induced hairy root responses. As shown in Fig. 5,
ATPgS induced a slightly lower medium pH increase and
a similar level of H2O2 production compared with those
produced by ATP; ADP induced much lower levels of
medium pH increase (Fig. 5A) and H2O2 production
(Fig. 5B) than those produced by ATP, and AMP induced
barely any response. These results confirmed that ATP
hydrolysis is not required for the ATP-induced responses in
the hairy root cultures. Fig. 5 also shows that eATP-induced
medium pH increase and H2O2 production were effectively
blocked by PPADS (pyridoxalphosphate-6-azophenyl-20 ,
40 -disulfonic acid), an alternative purinoceptor inhibitor to
the RB used above. In another complementary experiment,
the YE-induced H2O2 production was strongly suppressed
by PPADS and apyrase, the enzyme catalyzing the
hydrolysis of ATP (Fig. 6), providing further support for
the involvement of eATP in activating the YE elicitorinduced H2O2 biosynthesis. In a comparison of the results in
Figs. 6 and 2A, however, both apyrase (up to 25 U ml1)
and PPADS (at 100 mM) were less effective than RB
(at 40–100 mM) in blocking the YE-induced H2O2 production. According to previous studies (Chen et al. 1996,
Ralevic and Burnstock 1998), RB is a relatively potent
antagonist, and PPADS is a relatively weak and slow-action
antagonist of nucleotide receptors. The relatively weak
effect of apyrase on the YE-induced H2O2 was probably due
to the low dose applied.
Discussion
The above experimental results have shown that the
fungal elicitor YE can induce rapid and transient ATP
release from S. miltiorrhiza hairy roots, and the exogenous
ATP supplied to the culture can induce the early and
signaling events in plant cell response to biotic and abiotic
stress, Hþ influx and H2O2 production in the hairy root
cultures. Another interesting finding from the results is that
YE-induced H2O2 generation was dependent upon the
eATP binding to nucleotide receptors in the plasma
membrane, and was effectively blocked by the specific
inhibitor of nucleotide receptors RB. These findings provide
strong support for the signaling role of eATP in the
elicitation and activation of hairy root responses.
Note that the eATP inhibitor RB at sufficient doses
was able to block completely all the eATP-induced hairy
root responses, medium pH increase and H2O2 production
(Figs. 2, 4). This confirmed the efficacy of RB as an
antagonist for eATP signaling, and also proved the
important role of membrane nucleotide receptors for
perception and transmission of the eATP signal across the
plasma membrane of root cells. RB is a specific inhibitor of
purinoceptors found in the plasma membrane of mammalian cells (Song et al. 2006), though it could also be
inhibitory to some protein kinases. In addition to RB, the
eATP-induced responses were also suppressed by another
specific inhibitor of plasma membrane purinoceptors,
PPADS (Fig. 5). Therefore, our results here suggest the
existence of similar eATP purinoceptors in plant cells.
Extracellular ATP signaling in elicitor response
Moreover, the insignificant pH change and H2O2 production induced by AMP as shown in Fig. 5 also provide
support for the role of purinoceptors in mediating the ATPinduced responses as the specific purinoceptors (for ATP)
do not respond to AMP (Bours et al. 2006).
The effective blockage of the YE-induced H2O2
production by RB suggests that eATP is an important
signaling agent in the elicitation of ROS response in the
S. miltiorrhiza hairy roots. Based on the time courses shown
in Figs. 1 and 2, however, the YE-induced H2O2 was
detected earlier (10 min) than the ATP release. If the ATP
release was downstream of H2O2 production, how could
the eATP affect the YE-induced H2O2 production? One
possibility is that the eATP had a feed-back amplifying
effect on the earlier signaling events required for its own
release. Another possibility is that ATP was released into
the ECM or intercellular space at a much earlier time and
perhaps also at a much higher level than that detected in the
extracellular medium where the released ATP was highly
diluted (Jeter et al. 2004). According to Wu et al. (2007),
the ATP level on the membrane surface of mammalian
cells (lymphocytes) could be 1,000-fold higher than that
in the culture medium. Whether this also occurs in
plant cell cultures or in the actual eATP level in the
ECM of plant cells still needs to be confirmed through
621
direct measurement. As seen from our results shown in
Fig. 2A, a notable rise in the H2O2 level occurred within
10 min in the YE-treated culture, but did not occur until
after 30 min in the cultures treated with YE plus 40 or
100 mM RB. The significant effect of RB during this initial
period is suggestive of the release of ATP and an increase of
ATP at the cell surface to a sufficient quantity for a
significant effect on the elicitor-induced H2O2 production in
the early period. Song et al. (2006) suggested a threshold
eATP of 0.5 mM for the induction of ROS biosynthesis and
of 0.3 mM for the induction of cytosolic Ca2þ increase in
Arabidopsis leaves. At present, however, our experimental
results are still not sufficient to resolve whether eATP acts
as a signaling agent upstream or downstream of the H2O2
signal in the elicitor signal transduction.
As stated earlier in this report, ATP release from plant
cells may be attributed either to the loss of membrane
integrity or to some active transport means. The
YE-induced ATP release in the hairy root cultures should
be mainly caused by active transport means but not passive
means from membrane disruption, based on the two
following facts. The first is that the YE treatment of the
hairy root cultures caused no significant loss of cell viability
and membrane integrity in the hairy roots based on Evans
blue test as shown in Fig. 7A, and the second is that the
A
1
3
2
Root weight (g/flask)
B
Control
YE100 mg/L
2
1
0
fw
dw
Fig. 7 (A) Examination of cell viability and membrane integrity of hairy roots by Evans blue staining. (1) Healthy and intact roots from
normal shake-flask cultures as a positive control; (2) dead roots after 5 min in boiling water as a negative control; (3) roots treated with YE at
100 mg l1 for 10 h in shake-flasks. Hairy root samples were incubated with 0.25% Evans blue in MS medium for 10 min, and then rinsed
thoroughly with sterilized water. The root specimen was pressed into a thin layer and placed on a slide, observed and photographed under
a Leica DMBR microscope. The test was repeated three times and the microscopy field was chosen randomly over the specimen,
and representative photographss are shown in the figure. (B) Comparison of root weights in control and YE-treated hairy root cultures
(YE at 100 mg l1 and root weights measured 4 d after treatment in shake-flasks; fw, fresh weight; dw, dry weight).
622
Extracellular ATP signaling in elicitor response
YE-induced ATP release could be blocked by the Ca2þ
antagonists. On the other hand, the ATP release as a
consequence of cell growth observed in previous studies
(Kim et al. 2006, Wu et al. 2007) is unlikely to be a cause for
the YE-induced ATP here as the YE treatment had no
significant effect on the root growth (Fig. 7B). The strong
dependence of ATP release on the plasma membrane Ca2þ
influx also suggests that the elicitor-induced ATP secretion
is downstream of the Ca2þ influx and also mediated by
cytosolic Ca2þ. Kim et al. (2006) also found that the ATP
release in intact Medicago truncatula roots is a calciumdependent process. A possible route for the elicitor-induced
ATP release may be the calcium-mediated anion channels in
the plasma membrane, similar to those for Cl and NO
3
after elicitor treatment of plant cells (Ward et al. 1995,
Wendehenne et al. 2002). Such a route has been demonstrated for the ATP release in animal cells (Forrester 1990,
Abraham et al. 1993).
In contrast to the pH rise induced by exogenous ATP,
a notable pH drop or acidification of the hairy root medium
was induced by the elicitor YE. The medium pH change in a
cell culture is mainly caused by increasing Hþ influx into
(medium pH increase and alkalinization) or extrusion out of
the cells (pH drop and acidification). Proton extrusion by
membrane Hþ-ATPase provides the proton gradient across
the plasma membrane and is essential for maintaining the
membrane potential. The medium acidification may be a
result of Hþ-ATPase activation by the elicitor, leading to
excessive proton extrusion to the extracellular medium.
It has been suggested that specific elicitors bind to their
receptors in the plasma membrane, triggering the activation
of G-proteins, and the G-proteins transduce the signal by
activating the Hþ-ATPase, leading to hyperpolarization of
the membrane potential which induces the opening of a
Ca2þ channel and the membrane Ca2þ influx (Blumwald
et al. 1998). As Ca2þ influx was a prerequisite for the
YE-induced ATP release, the ATP release should be further
downstream of Hþ-ATPase activation and Hþ release or the
decrease in medium pH. Therefore, the insignificant effect
of eATP on the YE-induced pH change (decrease) may be
due to the fact that the YE-induced ATP release is
downstream of the pH decrease.
The strong dependence of eATP-induced medium pH
increase and H2O2 production as well as the YE-induced
ATP release on the membrane Ca2þ flux suggests that Ca2þ
plays a vital role in the eATP signal transduction and
eATP-induced responses in the hairy roots. On the other
hand, this may also suggest that eATP can act as a second
messenger in the Ca2þ-mediated elicitor signal transduction.
Our finding of the close association between eATP and
Ca2þ signaling is consistent with the results from previous
studies showing that eATP induces plasma membrane Ca2þ
flux and an increase in cytosolic Ca2þ level in plant cells
(Demidchik et al. 2003, Jeter et al. 2004), and the eATPinduced responses such as NADPH oxidase and ROS (O2–)
are dependent on Ca2þ (Song et al. 2006). Other relevant
findings from previous studies are that the ABC proteins
control the Ca2þ-regulated anion channels (Leonhardt et al.
1999), and that eATP and extracellular ADP depolarize the
plasma membrane of Arabidopsis root hairs (Lew and
Dearnaley 2000).
In conclusion, transient ATP release was an early event
in the response of S. miltiorrhiza hairy roots to a fungal
elicitor, and eATP played a significant role in the elicitor
signal transduction. The eATP signal was transmitted
across the plasma membrane via ATP-binding proteins
that are sensitive to RB and PPADS, similar to the
mammalian purinoceptors, and was closely linked to
plasma membrane Ca2þ influx. Further studies are needed
to map out the eATP signaling pathway and its interrelationship with other well-established signal elements in
the elicitor responses of plant cells.
Materials and Methods
Hairy root culture
The S. miltiorrhiza hairy root culture was derived after the
infection of plantlets with an Ri T-DNA-bearing Agrobacterium
rhizogenes (ATCC15834), maintained in a liquid, hormone-free
Murashige–Skoog (MS) medium with 30 g l1 sucrose but without
ammonium nitrate at 258C in the dark. The hairy root culture was
incubated in 125 ml Erlenmeyer flasks, each filled with 25 ml of
liquid medium, on an orbital shaker at 110–120 r.p.m. (shake-flask
cultures). Details of the hairy root culture have been given
elsewhere (Ge and Wu 2005).
Preparation of elicitor, exogenous ATP and inhibitor solutions
The elicitor YE was the polysaccharide fraction of yeast
extract obtained by ethanol precipitation, and the elicitor dose was
represented by the glucose-equivalent total carbohydrate content
as described previously (Ge and Wu 2005, Shi et al. 2007). As
shown in our previous studies, YE is a potent elicitor for
stimulating the tanshinone accumulation in S. miltiorrhiza hairy
root cultures. The most effective YE dose was shown to be about
100 mg l1 and was thus used in most of the experiments in this
study. The dependence of the elicitor response on eATP and Ca2þ
was examined through gain- and loss-of-function experiments by
applying their specific antagonists, i.e. RB as an inhibitor of eATP
signal transduction across the plasma membrane, and the Ca2þ
chelator EGTA and membrane channel blocker La3þ (with LaCl3)
as inhibitors of Ca2þ membrane influx. Additional control or
complementary experiments were conducted with a nonhydrolyzable form of ATP, ATPgS, and two hydrolyzed ATP
derivatives, ADP and AMP, to see whether they could mimic
the effects of ATP, and with another specific purinoceptor
inhibitor, PPADS, and apyrase, the enzyme for the hydrolysis of
ATP, to verify the effect of RB. YE, ATP, ATP derivatives and
the inhibitors were all pre-dissolved in distilled water as
100 concentrated stock solutions and sterilized by membrane
filtration. All these reagents were purchased from Sigma (St Louis,
MO, USA).
Extracellular ATP signaling in elicitor response
Elicitor, ATP and inhibitor treatments of hairy root cultures
The treatment experiments were carried out in 50 ml
Erlenmeyer flasks, each filled with 15 ml of fresh MS medium
and inoculated with 1.5 g FW of the hairy roots from the shakeflask cultures which had been incubated for 18–21 d. YE and ATP
were applied to the hairy root cultures at selected doses after an
initial incubation for 4 d. The eATP and Ca2þ inhibitors, when
needed, were added to the hairy root cultures 0.5 h before the
addition of YE and ATP.
All treatments were performed in triplicate flasks and
repeated at least once, and the results were represented as their
mean plus SD.
Measurement of ATP in the culture medium
ATP concentration in the culture medium was determined by
the luciferin–luciferase assay using a bioluminescence detection kit
(ENLITEN rLuciferin-Luciferase, Promega, Madison, WI, USA)
as reported by Jeter et al. (2004) and Wu et al. (2007). At selected
time intervals after various treatments, 100 ml of medium was taken
from each of the culture flasks and put into a sample tube, and
frozen immediately in liquid N2, and then stored in a 808C
refrigerator before the ATP assay. In the assay, the sample medium
was thawed at room temperature and mixed with 150 ml of Trisacetic acid buffer (100 mM, pH 7.8), and then with 50 ml of
the luciferin–luciferase reagent. The fluorescence intensity was
recorded on a TD-20/20 luminometer (Turner Designs, Sunny
Vale, CA, USA) with a reading delay of 2 s and an integration time
of 10 s. The fluorescence intensity was calibrated to the actual ATP
concentration with a pure ATP solution (Promega, Madison,
WI, USA).
Measurement of medium pH
The medium pH change after elicitor and ATP treatment is
an indication of the Hþ influx or efflux across the cell membrane.
The pH was measured after various treatments using an Orion
720Aþ pH meter with a pH electrode (Thermo Fisher Scientific,
Inc., Waltham, MA, USA). The culture flasks were shaken during
the measurement and care was taken to avoid perturbation of the
culture by the electrode.
Measurement of hydrogen peroxide
Hydrogen peroxide (H2O2) in the culture medium was
measured by luminol chemiluminescence as described by Wang
and Wu (2005) on the TD-20/20 luminometer. In brief, 50 ml of
sample medium was mixed with 750 ml of phosphate buffer
(0.05 M, pH 7.9), followed by auto-injection of 200 ml of luminol
(0.3 mM in phosphate buffer) and 100 ml of K3[Fe(CN)6] (14 mM in
water). Fluorescence intensity was recorded after the last injection
at an integration time of 5 s, and the intensity value was calibrated
to the actual H2O2 concentration with pure H2O2 liquid (30 wt% in
water from Junsei Chemical Co., Ltd, Tokyo, Japan).
Funding
The Hong Kong Polytechnic University (Nos. 1-BB80
and G-YF75).
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(Received January 13, 2008; Accepted February 20, 2008)