The Signaling Role of Extracellular ATP and its Dependence on Ca 2þ Flux in Elicitation of Salvia miltiorrhiza Hairy Root Cultures - PDF

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Plant Cell Physiol. 49(4): (8) doi:.93/pcp/pcn33, available online at ß The Author 8. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

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Plant Cell Physiol. 49(4): (8) doi:.93/pcp/pcn33, available online at ß The Author 8. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and the Japanese Society of Plant Physiologists are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact The Signaling Role of Extracellular ATP and its Dependence on Ca þ Flux in Elicitation of Salvia miltiorrhiza Hairy Root Cultures Shu-Jing Wu, Yuan-Shuai Liu and Jian-Yong Wu * Department of Applied iology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, PR China 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 mg l YE) at about h after YE treatment, but dropped to the control level 6 h later. The elicitor-induced ATP release was dependent on membrane Ca influx, and abolished by the Ca chelator EGTA or the channel blocker La 3. The YE-induced H O production was strongly inhibited by reactive blue (R), a specific inhibitor of membrane purinoceptors. On the other hand, the application of exogenous ATP at km to the cultures also induced rapid and dose-dependent increases in H O production and medium ph, both of which were effectively blocked by R 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 H O 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; Ca 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: Ca þ flux Extracellular ATP Fungal elicitor H þ flux H O production Salvia miltiorrhiza. Abbreviations: AC, ATP-binding cassette; eatp, extracellular ATP; ECM, extracellular matrix; MS, Murashige Skoog; PPADS, pyridoxalphosphate-6-azophenyl-, 4 -disulfonic acid; R, 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 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. 99, ours et al. 6). 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 Ca þ concentration (Shen et al. 5, ours et al. 6). 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. 3, Jeter et al. 4, Song et al. 6). 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 Ca þ concentration upon the application of exogenous ATP to Arabidopsis root or seedlings (Demidchik et al. 3). The increase in cytosolic Ca þ concentration due to plasma membrane influx or release from intracellular stores is an early event in plant response to biotic or abiotic elicitors, and Ca þ acts as a second messenger in the elicitor signal transduction (lumwald et al. 998, Zhao et al. 5). 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. 4, Song et al. 6). 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. 5) and Medicago truncatula root hairs (Kim et al. 6), and the stimulated ATP release by touch and osmotic stress in Arabidopsis seedlings (Jeter et al. 4). It has been suggested that the ATP release from plant cells may be accomplished by passive means resulting from plasma *Corresponding author: , Fax, þ 68 Extracellular ATP signaling in elicitor response membrane disruption, and by active means such as the ATP-binding cassette (AC) proteins through the anion channels, and the cell exocytosis of ATP-containing vesicles and subsequent vesicular fusion (Thomas et al., Kim et al. 6). 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. 5). 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 unge (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 99, Wang et al. 7). 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 993, Shi et al. 7). 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. Results YE-induced ATP release and dependence on Ca þ 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 3 min. The eatp concentration rose to a peak in about h, and then dropped back to the initial control level 6 h later (Fig. A). The eatp level increased with the YE dose, and the peak level at mg l was about 6.5 nm, which was 7 times higher than the control level. The YE-induced ATP release was completely blocked by both EGTA (5 mm) and La 3þ ( mm) (Fig. ). 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 Ca þ influx. A ATP (nm) ATP (nm) YE5 YE+La YEmg/l YE YE YE+EGTA Fig. Elicitor YE-induced ATP release in S. miltiorrhiza hairy root cultures (A); the inhibition of YE-induced ATP release by Ca þ antagonists () (EGTA at 5 mm and LaCl 3 at mm, added to the culture.5 h before YE addition). Error bars indicate the SD at n ¼ 3. YE- and eatp-induced H O production and dependence on Ca þ flux YE induced rapid production of H O in the hairy root cultures, leading to an increase in the H O concentration in the medium. The production of H O was detectable within min after YE addition to the culture, and reached the maximum level in about 4 h (Fig. A). The YE-induced H O production was effectively (nearly completely) blocked by the eatp inhibitor R ( mm) and completely blocked by the Ca þ chelator EGTA ( mm) (Fig. A), suggesting the strong dependence of YE elicitation of H O on both the eatp signal and the Ca þ influx across the plasma membrane. The rapid and transient production of ROS such as H O known as the oxidative burst is an early event in plant cell response to biotic and abiotic elicitors or stresses, and H O 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 H O production, detectable within min of the eatp supply (Fig. ). The H O concentration showed a small increase between and.5 h after the eatp supply, a period of slow and gradual increase between.5 and 3 h, and another notable increase to a peak at 4 h. Extracellular ATP signaling in elicitor response 69 A H O (µm) YE+EGTA YE+R YEmg/L YE+R4 Change of medium ph.... YE+EGTA EGTA YEmg/L YE+R YE+R5 3 ATP µm ATP4 µm ATP µm ATP4+R5 ATP4+EGTA Fig. 3 Elicitor YE-induced ph change (drop) in the hairy root culture medium and the effects of EGTA ( mm) and R ( and 5 mm). H O (µm) Fig. H O production induced by YE (A) and exogenous ATP (), and the inhibition by the Ca þ chelator EGTA and the eatp inhibitor R in the hairy root cultures. EGTA and R were added to the culture.5 h before YE; EGTA number is the dose in mm, R number is the dose in mm. The eatp-induced H O production in the hairy root cultures was completely inhibited by R ( mm) and EGTA ( mm) (Fig. ), suggesting its dependence on the membrane nucleotide receptors (blocked by R) and the Ca þ membrane influx (blocked by EGTA). YE- and eatp-induced ph change and dependence on Ca þ flux The elicitor YE caused a rapid and notable drop in the medium ph, by up to.5.3 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 Ca þ chelator EGTA or the eatp inhibitor R (Fig. 3). In contrast, the supply of eatp to the hairy root cultures caused a rapid increase in the medium ph, detectable within min of the eatp being supplied (Fig. 4A). The ATP-induced ph increase was most rapid and significant at mm, reaching a maximum of.7 ph units in 3 min (Fig. 4A). The eatp-induced ph increase was completely blocked by both EGTA at mm (Fig. 4A) and R at mm, and was partially blocked by R at lower doses of and 5 mm (Fig. 4). The results suggest the dependence of the eatp-induced ph increase A.4 Change of medium ph Change of medium ph.. ATP+R5 ATP+R ATP4 ATP+EGTA5 ATP ATP ATP+RµM R Fig. 4 Exogenous ATP-induced medium ph increase and the inhibition by EGTA and R in the hairy root cultures. ATP and R numbers are the doses in mm, and EGTA number is the dose in mm. on plasma membrane Ca þ influx and the membrane nucleotide receptors. The inhibitors EGTA and R alone also caused slight changes in the medium ph by no more than.3 units, which was negligible compared with that caused by YE or eatp. Effects of ATP derivatives, PPADS and apyrase on medium ph and H O production An additional control experiment was conducted with the non-hydrolyzable form of ATP, ATPgS, and two 6 Extracellular ATP signaling in elicitor response A.4.3 ATPγS AMP ATP ADP ATP+PPADS 5 4 YE+Apyase YE+PPADS YE mg/l YE+Apyase5 ph change. H O (µm) 3. H O (µm) Fig. 6 Effects of PPADS and apyrase on YE-induced H O production. Apyrase was supplied at or 5 U ml and PPADS at mm; PPDAS or apyrase alone induced no significant responses. antagonist, and PPADS is a relatively weak and slow-action antagonist of nucleotide receptors. The relatively weak effect of apyrase on the YE-induced H O was probably due to the low dose applied. Fig. 5 Medium ph change (A) and H O production () induced by the ATP analog and derivatives, and the inhibition of ATPinduced responses by PPADS. All reagents were supplied at 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 H O production compared with those produced by ATP; ADP induced much lower levels of medium ph increase (Fig. 5A) and H O production (Fig. 5) 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 H O production were effectively blocked by PPADS (pyridoxalphosphate-6-azophenyl-, 4 -disulfonic acid), an alternative purinoceptor inhibitor to the R used above. In another complementary experiment, the YE-induced H O 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 H O biosynthesis. In a comparison of the results in Figs. 6 and A, however, both apyrase (up to 5 U ml ) and PPADS (at mm) were less effective than R (at 4 mm) in blocking the YE-induced H O production. According to previous studies (Chen et al. 996, Ralevic and urnstock 998), R is a relatively potent 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, H þ influx and H O production in the hairy root cultures. Another interesting finding from the results is that YE-induced H O 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 R. 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 R at sufficient doses was able to block completely all the eatp-induced hairy root responses, medium ph increase and H O production (Figs., 4). This confirmed the efficacy of R 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. R is a specific inhibitor of purinoceptors found in the plasma membrane of mammalian cells (Song et al. 6), though it could also be inhibitory to some protein kinases. In addition to R, 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 6 Moreover, the insignificant ph change and H O 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 (ours et al. 6). The effective blockage of the YE-induced H O production by R suggests that eatp is an important signaling agent in the elicitation of ROS response in the S. miltiorrhiza hairy roots. ased on the time courses shown in Figs. and, however, the YE-induced H O was detected earlier ( min) than the ATP release. If the ATP release was downstream of H O production, how could the eatp affect the YE-induced H O 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. 4). According to Wu et al. (7), the ATP level on the membrane surface of mammalian cells (lymphocytes) could be,-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 direct measurement. As seen from our results shown in Fig. A, a notable rise in the H O level occurred within min in the YE-treated culture, but did not occur until after 3 min in the cultures treated with YE plus 4 or mm R. The significant effect of R 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 H O production in the early period. Song et al. (6) suggested a threshold eatp of.5 mm for the induction of ROS biosynthesis and of.3 mm for the induction of cytosolic Ca þ 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 H O 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 3 Root weight (g/flask) YE mg/l fw dw Fig. 7 (A) Examination of cell viability and membrane integrity of hairy roots by Evans blue staining. () Healthy and intact roots from normal shake-flask cultures as a positive control; () dead roots after 5 min in boiling water as a negative control; (3) roots treated with YE at mg l for h in shake-flasks. Hairy root samples were incubated with.5% Evans blue in MS medium for 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 DMR 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. () Comparison of root weights in control and YE-treated hairy root cultures (YE at mg l and root weights measured 4 d after treatment in shake-flasks; fw, fresh weight; dw, dry weight). 6 Extracellular ATP signaling in elicitor response YE-induced ATP release could be blocked by the Ca þ antagonists. On the other hand, the ATP release as a consequence of cell growth observed in previous studies (Kim et al. 6, Wu et al. 7) 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. 7). The strong dependence of ATP release on the plasma membrane Ca þ influx also suggests that the elicitor-induced ATP secretion is downstream of the Ca þ influx and also mediated by cytosolic Ca þ. Kim et al. (6) also found that the ATP release in intact Medicago truncatula roots is a calciumdependent process. A possible route for the elicitor-induc
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