Download Extracellular ATP in Plants. Visualization

Extracellular ATP in Plants. Visualization, Localization,
and Analysis of Physiological Significance in
Growth and Signaling1[W]
Sung-Yong Kim, Mayandi Sivaguru2, and Gary Stacey*
National Center for Soybean Biotechnology and Division of Plant Sciences (S.-Y.K., G.S.) and Division of
Biological Sciences and Molecular Cytology Core (M.S.), University of Missouri, Columbia, Missouri 65211
Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the
nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its
presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in
plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel
reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to
plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the
interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP
were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter
cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an
important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not
only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP
and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and
nonhydrolyzable ATP (bgmeATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased
ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.
ATP is an essential energy currency in nature and its
role in cellular metabolism is well established. The role
of extracellular ATP (eATP) as a signal molecule was
first noted in a 1929 study of the effects of adenine compounds on heart muscle contraction (Drury and SzentGyörgyi, 1929). Since then, a large body of literature
has documented the importance of eATP in a variety
of animal cell processes, including blood pressure regulation, immune responses, neurotransmission, and
cell growth regulation (Ralevic and Burnstock, 1998;
Burnstock and Williams, 2000). eATP in animals is
sensed through purinoceptors (plasma membrane
receptors). There are two classes of purinoceptors:
G-protein-coupled receptors (P2Y) and ligand-gated
ion channels (P2X). Purinoceptors affect intracellular
calcium signaling directly (P2X) or indirectly (P2Y;
Ralevic and Burnstock, 1998; Burnstock and Williams,
1
This work was supported by the National Research Initiative of
the U.S. Department of Agriculture Cooperative State Research,
Education and Extension Service (grant no. 2005–35319–16192).
2
Present address: Institute for Genomic Biology, University of
Illinois, 1207 West Gregory Dr., Urbana, IL 61801.
* Corresponding author; e-mail [email protected]; fax 573–
884–9676.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Gary Stacey ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.106.085670
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2000), resulting in a variety of downstream cellular
responses.
A possible role for eATP in plants was first reported
when exogenous application of ATP (100 mM) stimulated closure of the Venus fly trap (Jaffe, 1973) and an
excised Avena leaf showed increased formation of
nucleases (Udvardy and Farkas, 1973). Lew and
Dearnaley (2000) showed that exogenous ADP (10 mM)
and ATP (0.4 mM) depolarized the cell membrane
potential of growing Arabidopsis (Arabidopsis thaliana)
root hairs. Addition of ATP to plant roots was shown
to inhibit root gravitropism and polar auxin transport
(Tang et al., 2003). The presence of exogenous ATP
made plant roots more sensitive to auxin. Demidchik
et al. (2003) suggested a signaling role for ATP by
showing that its addition triggered an increase in
intracellular calcium levels. Thomas et al. (2000) suggested that plants could use the ATP gradient across
the plasma membrane to drive symport through a
multiple drug resistance transporter. Jeter et al. (2004)
showed that ATP-induced increases in cytoplasmic
calcium levels were coupled to downstream gene expression, implying a complete signaling pathway responsive to exogenous ATP. These authors suggested
that ATP released during plant wounding could be an
important signal molecule. This hypothesis was further strengthened by the recent results of Song et al.
(2006), who were able to extract cellular fluid from the
wound site and measured ATP levels up to 40 mM.
They also showed that ATP could trigger the formation
of reactive oxygen species (ROS). Therefore, ATP may
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Extracellular ATP in Plants
be an important plant signal molecule in response to
stress (e.g. wounding). More recently, Chivasa et al.
(2005) showed that Arabidopsis cells treated with
fumonisin B1, a programmed cell death-eliciting mycotoxin that activates plant defense, exhibited a death
response that was correlated with the depletion of
eATP. For example, a similar response could be seen by
adding enzymes that degraded ATP (e.g. apyrase) and
cell death could be reversed by adding exogenous
ATP. This article provided strong indirect evidence for
the presence of eATP in plants. The data presented by
Chivasa et al. (2005) suggest a more fundamental role
for eATP in the maintenance of cell viability.
The above studies clearly suggest that eATP may
exist in plants and could play a very important role in
cell viability and cellular signaling, similar to the
variety of functions assigned to eATP in animals. In
this article, we provide critical missing data regarding
the role of eATP in plants by visualizing eATP directly
using a novel reporter construct. The ability to visualize eATP and to provide relative quantification provided an opportunity to examine the mechanism of
eATP release using a variety of pharmacological agents.
The presence of eATP and the addition of exogenous
ATP correlated with increased production of ROS at
the tips of root hair cells. It is known that ROS production is critical to polar root hair growth (Foreman
et al., 2003) and correlated with a strong calcium
gradient (Wymer et al., 1997; Foreman et al., 2003).
Our data suggest that eATP is important for ROS
production and, therefore, may play a critical role in
polar root hair growth.
RESULTS
marker for imaging live root hairs or an indicator of
cellular activity. Treatment with the CBD-luciferase
reporter did not affect the viability of root hairs observed (Supplemental Movie S1).
Imaging of CBD-luciferase on the root surface revealed luciferase activity between epidermal cells,
suggesting release of eATP into the interstitial spaces
(Fig. 1E). Intense light was clearly seen at the root hair
tips (Fig. 1F). This pattern of eATP localization in
Medicago seems to be a general phenomenon because
Arabidopsis (ecotype Columbia-0; Fig. 1M), Triticum
aestivum (ET8; Fig. 1N), and Lotus japonicus (Gifu; Fig.
1O) also showed strongest fluorescence at the root hair
tip. Addition of exogenous ATP (Fig. 1G) or a chitin
mixture (Fig. 1H) resulted in strong fluorescence emission over the entire root hair surface. Application of
potato (Solanum tuberosum) apyrase (ATPase; Fig. 1I) or
nonhydrolyzable ATP (bgmeATP; Demidchik et al.,
2002; Fig. 1J) reduced fluorescence as compared to
control and exogenous ATP treatments. No fluorescence was detected in the absence of luciferin (Fig. 1, K
and L). The above results clearly demonstrate the
presence of eATP at the actively growing root surface
and root hair tips in a wide range of plants.
The level of eATP distribution at the root hair tips
was correlated with the location of the root hair in an
actively growing region (Fig. 2I). Young root hairs in
the most actively growing portion of the root showed
higher levels of eATP compared to older parts of the
root (Fig. 2I). After emergence, actively growing root
hairs and the root apex showed higher eATP. The use
of this reporter did not allow absolute quantification of
ATP levels in these experiments. However, relative
levels could be determined by photon counting or
intensity measurement (Fig. 3C).
ATP Is Most Abundant at the Root Hair Tip and Active
Growing Regions
CBD-Luciferase Protein Is Evenly Distributed at the Root
Hair Surface and Associated with the Cell Wall
To investigate whether eATP is present on the plant
root surface, a special reporter protein was constructed
by fusing a cellulose-binding domain (CBD) peptide
(Studier et al., 1990) with luciferase, an ATP-requiring
enzyme. The theory behind the use of the CBDluciferase fusion is that the CBD would anchor the
luciferase to the plant cell wall where it would sense
eATP. Fusion was expressed as a His-tagged protein in
Escherichia coli and affinity purified. Luciferase activity
of the protein was strictly dependent on the presence
of luciferin (Fig. 1A) and ATP (Moyer and Henderson,
1983; Fig. 1B). The CBD-luciferase reporter was applied to the roots of Medicago truncatula A17 (Jemalong) and luciferase activity in roots and root hair
tips was visualized in living cells/tissues either by
confocal microscopy (Theodossiou et al., 2003) or by a
deep-cooled CCD camera (ORCA AG; Hamamatzu
Photonics).
Figure 1, C and D, shows representative results from
imaging M. truncatula root hairs (see ‘‘Materials and
Methods’’). Cytoplasmic streaming was used as a
The intense fluorescence at the root hair tip may
reflect either higher eATP levels or could be due to an
uneven distribution of the CBD-luciferase reporter. To
confirm the binding of CBD-luciferase evenly to the
plant cell wall, roots were treated with the reporter
protein and the CBD was detected using immunofluorescence and immunogold labeling techniques. Figure 4
demonstrates that CBD-luciferase bound uniformly
over the entire surface of the plant root and hair
regardless of whether the primary antibody was directed against the CBD or the luciferase domain (Fig. 4,
B, D, E, J, and L). Figure 4J shows immunogold (10 nm)
labeling of the CBD, visualized using a gold enhancement technique, revealing uniform deposition of the
protein on the surface. The enhanced gold-gold complexes (relatively large in size) could be easily seen
under reflection mode in the confocal microscope; the
gold-gold enhancement technique does not enhance
all gold particles. Due to this reason, there are other
hard to see small gold particles (10 nm) between the
complexes in Figure 4J, which can be seen in Figure 4L,
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Kim et al.
Figure 1. Use of the CBD-luciferase reporter for
live imaging of eATP distribution on the root
surface of M. truncatula. A, Dose-response curve
for ATP showing light intensity was strictly dependent on ATP concentration. B, Light production was dependent on the presence of ATP and
substrate (luciferin). C, Projection of 36 confocal
optical sections (700 nm each) showing an overview of a live root segment demonstrating eATP
on several root hairs as revealed by CBDluciferase assay; raw data pseudocolored green.
D, Intensity-coded (inset range in D, where white
areas show the strongest ATP concentration and
black is the background) image of C showing ATP
concentration gradients. All other images are
single optical sections (700 nm thick). E, Section
of a root surface showing increased eATP secretion in the interstitial spaces of epidermal cells. F,
Medicago truncatula (A17) root hair showing light
production primarily at the root hair tip. CBDluciferase light production was greatly stimulated
by the addition of exogenous ATP (100 mM; G) and
by chitin elicitation (100 mg/mL chitin mixtures;
H). Light production was decreased by the addition of potato apyrase (ATPase; 25 units/mL; 1 unit
will liberate 1 mM of inorganic phosphate from
ATP or ADP per min at pH 6.5 at 30°C; I) or
100 mM nonhydrolyzable ATP (bgmeATP; J). A
negative control (i.e. without added luciferin)
showing that there was no autofluorescence in
root hairs in the absence (K) or presence (L) of
CBD-luciferase treatment. Light production upon
CBD-luciferase treatment was seen in the root
hairs of a variety of plants. M, Arabidopsis
(Columbia-0). N, T. aestivum (ET8). O, L. japonicus (Gifu). Light production in Figure 1, A and B,
was measured using a luminometer (Veritas microplate luminometer; Turner BioSystem). All root
hairs presented are captured in and around region
B in Figure 5I. Scale bar 5 10 mM.
suggesting uniform distribution of the fusion protein
along the root hair surface and interstitial spaces of the
roots (Fig. 4E). Figure 5II shows fluorescence from a
root hair that was plasmolyzed by treatment with 5 M
NaCl, resulting in shrinkage of the plasma membrane,
making it retract from the cell wall (Fig. 5IIB and the
overlay, Fig. 5IIC). These images clearly illustrate that
CBD-luciferase fluorescence is associated predominantly with the cell wall and, therefore, eATP localization is extracellular.
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Extracellular ATP in Plants
Figure 2. ATP distribution profile in M. truncatula root and enhanced eATP secretion is restricted to actively growing regions of
the plant. I, All images are single confocal optical sections (700 nm). M. truncatula root (left, bright-field image) was divided into
three sections designated as A, B, and C. The root apex is marked with an asterisk (*). Representative images showing light
production from CBD-luciferase are shown (right) and correspond to the three regions. White areas show the strongest eATP
concentration and black is the background (see scale; Fig. 1). II, CBD-luciferase activity was detected at interstitial spaces at
different growth regions of the root (i.e. meristematic region [A and B], apical elongation region [C], basal elongation region [D],
mature region [E], and epidermal cells of the etiolated hypocotyl [F]). White boxes were drawn on the images to highlight the
areas of interstitial spaces between epidermal cells. Scale bar 5 25 mM.
ATP Secretion Is Dependent on Calcium
The distribution pattern of ATP shown in Figure 6A
suggests that eATP release may occur as part of the
normal, polar growth of the root hair (Kropf et al.,
1998). Calcium influx and establishment of a cytoplasmic calcium gradient at the root hair tip is essential for
root hair elongation (Schiefelbein et al., 1992). In animal
cells, an increase in cytoplasmic calcium is a downstream consequence of eATP (Dubyak and el-Moatassim,
1993; Leipziger, 2003; Shaw and Long, 2003). Therefore,
CBD-luciferase activity was assayed in the presence of
known inhibitors of calcium influx (Jeter et al., 2004;
e.g. GdCl3, LaCl3; Fig. 5I, B and C; see Fig. 3A for
quantification), calcium chelators [Jeter et al., 2004; e.g.
1,2-bis(o-aminophenoxy)ethane-N,N,N#,N#-tetraacetic
acid (BAPTA); Figs. 5ID and 3A], or elevated exogenous
CaCl2 (5 mM; Figs. 5IE and 3A). Inhibition of calcium
influx or chelation of calcium greatly reduced the
presence of eATP. In contrast, elevated levels of eATP
were seen upon addition of exogenous calcium.
One mode of ATP release in animal cells is through
exocytotic release of ATP as vesicles fuse with the
plasma membrane (Bodin and Burnstock, 2001; Bowler
et al., 2001). Vesicles also fuse with the tip of the
growing root hair as part of the polar growth process
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Kim et al.
(Battey et al., 1999; Shaw et al., 2004). M. truncatula
roots were treated with brefeldin A, a well-characterized
inhibitor of vesicular trafficking (Klausner et al., 1992;
Molendijk et al., 2001) and subsequently imaged after
addition of the CBD-luciferase reporter. This treatment
strongly reduced the presence of eATP (Figs. 5IF and 3A),
again demonstrating the importance of polar growth
processes for the release of ATP in plant root hairs.
ATP Increases ROS Activity at the Root Hair Tip
Analysis of ROS distribution showed strong activity at the actively growing region of the root hair tip
(Fig. 6IA). There was no change in control ROS activity,
Figure 3. Quantification of eATP and ROS intensity of M. truncatula. A,
Quantification of ATP based on light intensity for the images presented
in Figures 1 and 5I. Images were analyzed for intensity average in the
Metamorph program, version 6.5 (Molecular Devices). Measurements
were made at the root apices and if any contrast adjustments were
needed, the adjustments were applied to all root hairs prior to analysis.
Values (n . 8) are mean 6 SE in an experiment and representative of at
least two independent experiments. B, Quantification of ROS production based on intensity for the images presented in Figure 6I. Images
were analyzed for intensity (average) in the Metamorph program,
version 6.5. Measurements were made at the root apices. Whenever
contrast adjustments were needed, the adjustments were applied to all
root hairs prior to analysis. Values (n . 8) are mean 6 SE in an
experiment and representative of at least two independent experiments.
C, Quantification of ATP based on intensity for the images presented in
Figure 2I. A (proximal), B (middle), and C (distal) refer to the three root
regions shown in Figure 2I. Images were analyzed for intensity average/
sum in the Metamorph program, version 6.5. Measurements were made
at the root hair apices. Whenever contrast adjustments were needed,
the adjustments were applied to all root hairs identically prior to
analysis. Values (n . 8) are mean 6 SE in an experiment and representative of at least two independent experiments.
Figure 4. Immunofluorescence and immunogold labeling of CBDluciferase protein showing uniform localization around the root hair
and interstitial spaces of the root surface. A to F, Images are after
fluorescent antibody (Ab) labeling. CBD-luciferase protein was localized using a mouse monoclonal antibody followed by Alexa fluor 568
conjugated secondary antibody on M. truncatula roots pretreated with
CBD-luciferase protein. Root hair incubated with CBD preimmune
serum and secondary antibody (A), control root hair incubated with
both primary (anti-CBD) and secondary antibody (B), root hair incubated with luciferase preimmune serum (C and F), and root hair
incubated with both primary (anti-luciferase) and secondary antibody
(D and E). E and F, From the elongation region of the root apex
(projection of 17 confocal optical sections, 700 nm each). All fluorescent images are single confocal optical sections (700 nm), except E and
F. G to L, Images are after immunogold (10-nm gold particles) labeling
with gold enhancement after binding of luciferase preimmune antibody
or primary (anti-luciferase) antibody and obtained in a confocal microscope under reflection mode. G, I, and K are transmitted images of
H, J, and K. Root hair incubated with luciferase preimmune serum and
secondary antibody (H) and primary (anti-luciferase) antibody and
secondary antibody (J and L). All root hairs presented are captured
in and around region B in Figure 2I. Scale bar in A to D and G to
L 5 500 mM; E and F 5 10 mM.
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Extracellular ATP in Plants
root hairs grew at the rate of 1.8 mM 6 6.9 min21
compared to the root hairs supplied with 25 units/
mL potato apyrase (ATPase) with an average growth
rate of 0.016 mM 6 0.197 min21 (Supplemental Movie
S2). These results further indicate that eATP is essential for normal growth of the root hair cell, perhaps
through the production of ROS.
Enhanced eATP Secretion Is Restricted to Actively
Growing Regions of Plant Cells
Figure 5. ATP secretion is dependent on cytoplasmic calcium and ATP
distribution is extracellular and localized at the plant cell wall. I, All
images are single confocal optical sections (700 nm). A, Optical section
of a root hair treated with CBD-luciferase showing eATP distribution.
Similar root hairs treated with pharmacological agents for 1 h prior to
CBD-luciferase application are shown in B (GdCl3), C (LaCl3), D
(BAPTA), E (CaCl2), and F (brefeldin A). See Fig. 3A for quantification of
these treatments. Intensity-coding strip shown in A, where white areas
show the strongest eATP concentration and black is the background.
Scale bar 5 10 mM. II, Optical section showing eATP (CBD-luciferase
light activity) distribution on the root hair surface after plasmolysis with
5 M NaCl (30 min; A); transmission image of the same root hair showing
the plasma membrane is retracted from the cell wall (B); and overlay of
A and B, showing that light production was associated with the cell wall
(C). All root hairs presented are captured in and around region B in
Figure 3I. Scale bar 5 500 mM.
which remained at basal levels for at least 15 min (Fig.
6II). Addition of exogenous ATP increased ROS activity that subsequently and gradually decreased to basal
levels after 15 min (Fig. 6II). However, exogenous
application of nucleotides such as adenosine, AMP,
ADP, and nonhydrolyzable ATP (bgmeATP) did not
change ROS activity (Fig. 6I, C–F; see Fig. 3B for quantification). However, exogenous application of potato
apyrase (ATPase) decreased ROS activity (Figs. 6IH
and 3B). Application of an exogenous chitin mixture, a
strong elicitor of plant defenses, led to higher ROS
activity than that of the control with expression over
the entire root hair surface (Figs. 6IG and 3B). Chitin
elicitation mimicked the application of exogenous
ATP, calcium, and the chitin mixture by inducing
increased eATP (Figs. 1, G and H, and 5IE). The strong
production of ROS upon chitin addition was reversed
when chitin was added in conjunction with potato apyrase (ATPase; Figs. 6I and 3B). The induction of ROS
and the colocalization of eATP in root hairs suggests a
mechanistic connection and points to an important
role for eATP in polar root hair growth.
To further address the physiological significance of
eATP for root hair growth, we examined root hair
growth using time lapse photography in the presence
and absence of potato apyrase (ATPase). The control
Analysis of eATP secretion along the root apex at
different growth zones and in the etiolated hypocotyl
showed differing degrees of eATP secretion along the
interstitial spaces (Fig. 1E), depending on the growth
status of the cells. In these cases, cell expansion is
perpendicular to the axis of the root. The highest
growth is expected along the longitudinal walls of the
Figure 6. ROS profile and a time course analysis of ROS production in
root hairs. I, All images are single confocal optical sections (700 nm).
Optical section of control root hair treated with CM-H2DCFDA showing ROS fluorescence (A); similar root hair applied with 1 mM ATP (B);
1 mM nonhydrolyzable ATP (bgmeATP; C); 1 mM adenosine (D); 1 mM
AMP (E); 1 mM ADP (F); and 100 mg/mL chitin mixture (G). H,
Exogenous potato apyrase (ATPase; 25 units/mL; 1 unit will liberate
1 mM of inorganic phosphate from ATP or ADP per min at pH 6.5 at
30°C) decreased ROS levels. I, Potato apyrase was applied after chitin
treatment (30 min). There was also no fluorescence in the ROS negative
control roots (J; i.e. in the absence of the CM-H2DCFMA indicator dye),
compared to control (A). Scale bar 5 500 mM. II, There was no change
in control (top, without added ATP) ROS levels and basal levels were
maintained for at least 15 min. Addition of exogenous ATP increased
ROS activity that gradually decreased to a basal level within 15 min
(bottom). All root hairs presented are captured in and around region B
in Figure 3I.
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Kim et al.
epidermal and cortical cells in the etiolated hypocotyl,
which showed the highest eATP levels (Fig. 3IIF). On
the other hand, in the case of the root apex in the
meristematic zone, cell expansion is minimal and,
accordingly, eATP levels were also minimal (Fig. 3II,
A and B). As expected, eATP levels were higher in the
elongation zone of the same root apex (Fig. 3II, C and
D) and showed a decreasing trend again toward the
more mature regions of the root (Fig. 3IIE). These
results clearly indicate there is a direct correlation
between eATP secretion levels and cellular expansion/
growth status of cells.
DISCUSSION
Cell division and elongation are important processes during plant development. Root and root hair
growth has been well studied due to its dynamic
nature (more than 1 mM/min) and ease by which the
process can be visualized. Root hairs arise from root
epidermal trichoblast cells (located outside an anticlinal cortical cell wall). Root hairs develop from trichoblasts as a small bulge and expand by polarized
deposition of cell wall material (Miller et al., 1997).
Secretory vesicles, attached to cytoskeletal elements,
carry the cell wall materials (pectins, hemicelluose,
and glycoproteins) to the plasma membrane and polar
growth occurs at the root hair tip (Bibikova et al.,
1997). A strong, tip-oriented calcium gradient exists in
the root hair. Addition of verapamil, a calcium channel
blocker, inhibited root hair tip growth without affecting root hair initiation (Wymer et al., 1997). Production
of ROS is essential for polar root hair growth. Root
hairs under normal growth conditions showed strong
ROS activity and this could be blocked by diphenylene
iodonium, an inhibitor of NADPH oxidase (Foreman
et al., 2003). In line with this, the Arabidopsis root
hair defective mutant (rhd2), defective in NADPH
oxidase, showed decreased ROS activity and inhibited
calcium uptake at the root hair tip (Foreman et al., 2003).
This mutant is blocked in root hair development at the
stage at which trichoblast cells exhibit the initial bulge.
Root hair tip growth is dependent on the cytoplasmic calcium gradient and vesicular trafficking. Increasing the root hair cytoplasmic calcium gradient
from basal levels stimulated exocytosis and root hair
cell elongation (Cramer and Jones, 1996; Carroll et al.,
1998). When exogenous calcium was added at the end
of the root hair tip, the root hair growing direction was
changed (Bibikova et al., 1997). These data suggest that
the cytoplasmic calcium gradient, likely through action on the cytoskeleton, was critical to determining
the direction of polar growth. This might have biological relevance to root hair infection by nitrogen-fixing
rhizobia, where exogenous addition of the lipochitin
nodulation signal, produced by the symbiont, modulated cytoplasmic calcium levels and led to a change in
the direction of root hair growth (Felle et al., 1999;
Esseling et al., 2003; Oldroyd and Downie, 2004).
McAlvin and Stacey (2005) reported that transgenic
expression of soybean (Glycine max) GS52 apyrase in L.
japonicus resulted in a doubling of nodule number and
a concomitant increase in root infections by Mesorhizobium loti. GS52 is an ectoapyrase with the ATPase
catalytic domain predicted to be extracellular. These
data suggest that hydrolysis of eATP by apyrase was
beneficial to rhizobial infection. Exogenous addition of
the Nod signal increases cytoplasmic calcium while
decreasing ROS activity (Shaw and Long, 2003). Therefore, it is interesting to speculate that apyrase may
reduce eATP levels, resulting in lower ROS activity
during rhizobial infection. This may be a mechanism
for the symbiont to avoid defense reactions that may
be triggered by elevated ROS.
The use of CBD-luciferase allowed visualization of
eATP in living cells. This system was validated by a
variety of methods to confirm that CBD-luciferase
targeted the extracellular cell wall space and was
strictly dependent on the presence of plant-produced
eATP (Figs. 1 and 3). Further, analysis of this eATP
secretion and its physiological significance in terms of
actively growing root hairs, root zones, and etiolated
hypocotyl regions clearly indicates that eATP secretion
is inherently associated with the growth status of
various cell types (Figs. 1 and 3). Slower growing
root hairs in mature regions of the roots and trichoblasts that showed only an initial bulge had lower
levels of polar-localized eATP (Fig. 3). The presence of
polar eATP on plant root hairs appears to be a general
phenomenon and was seen with Medicago, Arabidopsis, Triticum, and Lotus root hairs (Fig. 1).
Secretion of eATP was dependent on calcium, as
evidenced using a variety of pharmacological agents
(Fig. 5) and ionophores (i.e. ionomycin and A23187;
data not shown). We postulate that this response is
due to the effect of calcium on vesicular transport and
exocytosis at the root hair tip (Carroll et al., 1998;
Demidchik et al., 2002). Consistent with this hypothesis, addition of brefeldin A, an inhibitor of vesicular
transport, blocked eATP release (Fig. 5I). Such a mechanism would closely resemble the release of eATP that
occurs at the animal nerve synapse where eATP acts as
an important neurotransmitter (Edwards et al., 1992).
However, the precise molecular mechanism of eATP
release awaits further investigation.
eATP, as measured using CBD-luciferase, colocalized at the root hair tip with regions of high ROS
activity (Fig. 6I). These two processes are likely mechanistically linked because the addition of exogenous
ATP greatly increased ROS production (Fig. 6IB).
Moreover, chitin, a known elicitor of plant defense
responses, elicited high ROS activity that was correlated with increased eATP (Figs. 1H and 6IG). Although we cannot rule out the possibility that a chitin
mixture could have affected the binding characteristics
of the fusion protein, this seems unlikely given the
binding specificity and affinity of the CBD.
A variety of earlier publications suggested a role for
eATP in plant cell signaling, but such a role is only
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Extracellular ATP in Plants
possible if eATP is normally present in living plant
cells. Our results provide this missing link by directly
visualizing eATP in actively growing plant cells. The
data are consistent with a critical role for eATP in the
growth of plant cells. Localization of eATP correlated
with regions of plant cell expansion. Production of ROS
is known to promote cell wall loosening, required for
cell expansion (Passardi et al., 2005). Therefore, eATP
may serve as a growth signal, triggering cell wall expansion via ROS production. Future research should
focus on how plants recognize eATP. Song et al. (2006)
recently reported that pharmacological agents known
to inhibit animal P2 ATP receptors abolished ATPdependent ROS production on Arabidopsis leaves,
suggesting that such receptors may exist in plants.
However, our database searches for genes showing
significant similarity to the two known families of
animal purinoceptors did not reveal any obvious candidates.
MATERIALS AND METHODS
Plant Germination and Growing Conditions
Medicago truncatula A17 (Jemalong) seeds were sterilized with concentrated sulfuric acid for 10 min, rinsed with sterilized water, soaked in 5%
commercial bleach for 3 min, and rinsed three times with sterilized water.
Seeds were stored at 4°C for 48 h and germinated with roots hanging down
from the roof of petri dishes. Seedlings were placed in an incubator (25°C) for
5 d prior to use.
cases, confocal optical sections of 0.5 to 1.0 mm were obtained all along the root
hair, including the surface, to reconstruct the three-dimensional image and
plane-by-plane analysis of signal intensity. Most presented images were a
single optical section along the median plane of the root hair. All images were
processed identically to clear the detector noise improving the signal-to-noise
ratio using Metamorph image analysis software, version 6.0 (Molecular
Devices). Quantitative confocal microscopy analysis of the signal intensities
was performed using the same software. Statistical significance was evaluated
with Student’s t test.
Immunofluorescence and Immunogold Detection of the
CBD and Luciferase Domain
Whole-mount immunofluorescence preparations and antibody staining of
M. truncatula were performed essentially as described (Sivaguru et al., 2003).
Briefly, root segments were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde, washed, extracted in 100% ice-cold (220°C) methanol, rehydrated,
blocked with goat serum, and incubated in either 1:200 monoclonal anti-CBD
antibody produced in mouse clone CBD-8 or monoclonal luciferase antibody
produced in mouse clone LUC-1 (Sigma-Aldrich), diluted in blocking buffer
(2% IgG-free goat serum, 2% IgG-free bovine serum albumin, and 0.1% MicroO-Protect), and incubated overnight. After washing, samples were incubated
in an anti-mouse secondary antibody conjugated with Alexa fluor 568
(Invitrogen), diluted 1:500 in blocking buffer, subsequently mounted in an
antifade mounting medium (Mowiol; Calbiochem), and imaged under the
confocal microscope using a 568-nm excitation line (600/40 emission) of a Kr/
Ar mixed-gas laser with the objectives, optimum iris/pinhole, and settings as
described above. Alternatively, the samples labeled with monoclonal antiluciferase antibody were processed with another secondary anti-mouse antibody conjugated with 10 nm gold (Aurion), either imaged directly or after
gold enhancement (Nanoprobes) according to the manufacturer’s protocol
either in the same confocal microscope using a polarization filter in the path
of a 488-nm Kr/Ar laser line (reflection mode) or under a scanning electron
microscope.
Treatment of M. truncatula with Pharmacological Agents
CBD-Luciferase Construct
The firefly (Photinus pyralis) luciferase gene from pJD301 (Luehrsen et al.,
1992; kindly provided by Dr. Albrecht von Arnim, University of Tennessee,
Knoxville) was ligated into the NcoI-SacI site of pET-36b(1) (Novagen; EMB
Bioscience). The CBD-luciferase construct was expressed in Escherichia coli
BL21(DE3) pLysS (Novagen; EMB Bioscience) and subsequently purified by
affinity chromatography using a Novagen CBind and/or S-tag purification kit
following the manufacturer’s instructions. Luciferase activity was checked
using the luciferase fluorescence assay system from Promega after CBDluciferase protein purification.
Imaging CBD-Luciferase Using Confocal Microscopy
Approximately 5 mL (100 mg/mL) CBD-luciferase protein solution was
added to the root system of a 5-d-old M. truncatula seedling. The M. trucatula
A17 seedling was incubated with the reporter, without rocking, for 1 h at room
temperature. The seedling was then washed three times with sterile water. The
luciferase-produced light was imaged with an inverted Olympus IX 70
microscope (Olympus) coupled with a Bio-Rad-Radiance 2000 laser-scanning
confocal system (Zeiss) by addition of 200 mL flash assay buffer (20 mM Tricine),
2.67 mM MgSO4, 0.1 mM EDTA, 2 mM dithiothreitol, and 470 mM D-luciferin
(Sigma-Aldrich; van Leeuwen et al., 2000). Using either a UV excitation
(350 nm) source (mercury lamp at 100 W) or the 488-nm argon laser with a
minimum power setting, the light signal from the samples was captured in
complete darkness using a 60 3 Olympus water immersion objective (Uplanapochromat; 1.2 NA) with a long-range emission filter (500 LP), optimum
gain, and the pinhole/iris wide-open. Images were captured using the
confocal photmultiplier tube in grayscale and stored to hard discs. Alternatively, results were compared using the same setup because the images were
also captured in a deep-cooled CCD camera (ORCA AG; Hamamatzu Photonics). All settings were optimized for live cell imaging and constant
parameters were used within a day across experimental treatments to compare signal intensity. No light was detected from plants treated with CBDluciferase without the substrate luciferin present (Fig. 1, K and L). In some
Nonhydrolyzable ATP (bgmeATP), potato (Solanum tuberosum) apyrase
(ATPase), GdCl3, BAPTA, LaCl3, and brefeldin A were purchased from SigmaAldrich. M. truncatula seedlings were pretreated with each of the agents in
water for 1 h at room temperature. All reagents were prepared fresh at the
following concentrations: nonhydrolyzable ATP (bgmeATP; 1 mM), potato
apyrase (ATPase; 25 units/mL), GdCl3 (1 mM), LaCl3 (1 mM), BAPTA (10 mM),
and brefeldin A (25 mg/mL). After treatment, seedlings were washed three
times with sterile water prior to treatment with CBD-luciferase as described
above.
Imaging ROS Using Confocal Microscopy
Seedlings were incubated at room temperature for 30 min with 5 to 20 mM
CM-H2DCFDA (Molecular Probes; Invitrogen), in B&D liquid medium (pH
7.0; Broughton and Dilworth, 1971). Labeled seedlings were washed with
B&D medium and left for 30 min at room temperature before the experiment.
A confocal microscope was used as described above. Briefly, the 488-nm
excitation light of the Kr/Ar laser was used to excite the CM-H2DCFDA and
the light signal from samples was captured under a 60 3 Olympus water
immersion objective (Uplanapochromat; 1.2 NA) with a green band-pass
emission filter (515/30). Laser power, gain, and other parameters were set
ideal for live cell imaging. For elicitor experiments, Medicago seedlings were
treated with crab shell chitin (a chitin mixture; Sigma-Aldrich) for 30 min at a
final concentration of 100 mg/mL (Wan et al., 2004).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Movie S1. Root hair viability is not affected while imaging
eATP secretion as indicated by cytoplasmic streaming.
Supplemental Movie S2. Exogenous potato apyrase (ATPase) inhibits
root hair growth in M. truncatula.
Plant Physiol. Vol. 142, 2006
991
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2006 American Society of Plant Biologists. All rights reserved.
Kim et al.
ACKNOWLEDGMENT
We thank the staff at the Molecular Cytology Core for all imaging
techniques in confocal microscopy and image processing.
Received June 22, 2006; accepted August 28, 2006; published September 8,
2006.
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