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DEVELOPMENT AND HORMONE ACTION

Extracellular ATP in Plants. Visualization, Localization, and Analysis of Physiological Significance in Growth and Signaling^1,[W]

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

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 (meATP). 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.

A possible role for eATP in plants was first reported when exogenous application of ATP (100 µM) 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 µM) 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 µM. They also showed that ATP could trigger the formation of reactive oxygen species (ROS). Therefore, ATP may 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

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

ATP Is Most Abundant at the Root Hair Tip and Active Growing Regions

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 CBD-luciferase 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).

View larger version (58K): [in this window] [in a new window]   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 CBD-luciferase 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. CBD-luciferase light production was greatly stimulated by the addition of exogenous ATP (100 µM; G) and by chitin elicitation (100 µg/mL chitin mixtures; H). Light production was decreased by the addition of potato apyrase (ATPase; 25 units/mL; 1 unit will liberate 1 µM of inorganic phosphate from ATP or ADP per min at pH 6.5 at 30°C; I) or 100 µM nonhydrolyzable ATP (meATP; 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 = 10 µM.

View larger version (83K): [in this window] [in a new window]   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 = 10 µM. 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 = 500 µM.

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 (meATP; 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 ).

View larger version (120K): [in this window] [in a new window]   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 = 25 µM.

View larger version (18K): [in this window] [in a new window]   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 ± 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 ± 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 ± SE in an experiment and representative of at least two independent experiments.

View larger version (109K): [in this window] [in a new window]   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 (meATP; C); 1 mM adenosine (D); 1 mM AMP (E); 1 mM ADP (F); and 100 µg/mL chitin mixture (G). H, Exogenous potato apyrase (ATPase; 25 units/mL; 1 unit will liberate 1 µM 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 = 500 µM. 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.

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, 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.

View larger version (46K): [in this window] [in a new window]   Figure 4. Immunofluorescence and immunogold labeling of CBD-luciferase 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 = 500 µM; E and F = 10 µM.

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 (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, 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 (meATP) 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 root hairs grew at the rate of 1.8 µM ± 6.9 min^–1 compared to the root hairs supplied with 25 units/mL potato apyrase (ATPase) with an average growth rate of 0.016 µM ± 0.197 min^–1 (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

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 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

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 µM/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 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 ATP-dependent 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

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

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.

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(+) (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 CBD-luciferase protein purification.

Imaging CBD-Luciferase Using Confocal Microscopy

Approximately 5 mL (100 µg/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 µL flash assay buffer (20 mM Tricine), 2.67 mM MgSO₄, 0.1 mM EDTA, 2 mM dithiothreitol, and 470 µM 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 60x 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 CBD-luciferase without the substrate luciferin present (Fig. 1, K and L). In some cases, confocal optical sections of 0.5 to 1.0 µm 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 (–20°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% Micro-O-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 anti-luciferase 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

Nonhydrolyzable ATP (meATP), potato (Solanum tuberosum) apyrase (ATPase), GdCl₃, BAPTA, LaCl₃, and brefeldin A were purchased from Sigma-Aldrich. 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 (meATP; 1 mM), potato apyrase (ATPase; 25 units/mL), GdCl₃ (1 mM), LaCl₃ (1 mM), BAPTA (10 mM), and brefeldin A (25 µg/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 µM 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 60x 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 µg/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.

	ACKNOWLEDGMENTS

 
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.

	FOOTNOTES

 
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.

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 (staceyg{at}missouri.edu).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.085670

^* Corresponding author; e-mail staceyg{at}missouri.edu; fax 573–884–9676.

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