JXB Advance Access originally published online on October 24, 2005
Journal of Experimental Botany 2005 56(422):3129-3136; doi:10.1093/jxb/eri310
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RESEARCH PAPER |
Harpin modulates the accumulation of salicylic acid by Arabidopsis cells via apoplastic alkalization
Institute of Biological Sciences, University of Wales, Aberystwyth, Edward Llwyd Building, Penglais Campus, Aberystwyth SY23 3DA, Wales, UK
* To whom correspondence should be addressed. Fax: +44 (0)1970 622311. E-mail: rmd{at}aber.ac.uk
Received 7 April 2005; Accepted 8 September 2005
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Abstract |
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It is reported here that salicylic acid (SA) is rapidly taken up by Arabidopsis cells, and its uptake is accompanied by media alkalization and cytosolic acidification, and it is inhibited by the ionophore nigericin, suggesting that its import is linked with that of H+ and driven by a proton gradient. Such import and accumulation declined sharply within a narrow physiological pH range (pH 5.7–6.1), corresponding to a reduction in the [H+] of the media from 1.99 µmol l–1 to 0.79 µmol l–1. Following the initial uptake, SA was exported back into the media as free SA against a continued [H+]-dependent import. Since the uptake and accumulation of SA declines sharply within a narrow pH range and cell wall alkalization is an early response during incompatible plant/pathogen interactions, the bacterial elicitor harpinPss was used to investigate how SA transport may be modulated during defence responses. Harpin induced a rapid and sustained alkalization of the cell suspension media, reaching the critical pH (pH 5.9–6.1) at which SA import is inhibited at c. 60 min. Such media alkalization corresponded with a reduction in the SA associated with cells co-treated with harpin, and an inhibition of SA uptake in cells pretreated with harpin. Scavengers of ROS, or compounds which generate H2O2 or NO had little effect on the import or net export of SA, suggesting that media alkalization induced by harpin is sufficient to modulate the kinetics of SA transport.
Key words: Apoplastic alkalization, Arabidopsis thaliana, harpin, nitric oxide, proton gradient, reactive oxygen species, salicylic acid
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Salicylic acid (SA) is a key regulator of plant defences, both in the enhancement of local defence responses and the establishment of the broad-based systemic acquired resistance (SAR; Mauch-Mani and Métraux, 1998
). Its production at the site of infection has been linked with the induction of defence-related gene expression, the enhanced generation of reactive oxygen species (ROS) and programmed cell death (PCD, Mur et al., 1996; Shirasu et al., 1997). Although subsequent systemic SA accumulation is essential for SAR, this may not be mobilized from the site of infection (Malamy et al., 1990; Métraux et al., 1990; Gaffney et al., 1993; Summermatters et al., 1995). Studies have demonstrated SA in the phloem exudates of cucumber following infection (Métraux et al., 1990), while feeding labelled SA precursors to infected leaves resulted in labelled SA accumulating in uninfected leaves (Mölders et al., 1996). However, grafting experiments using tobacco plants expressing the bacterial hydroxylase gene (nahG) and wild-type plants suggested that, whilst systemic SA accumulation was necessary for the induction of SAR, the SA was synthesized in situ and transport from the infected site was not required (Vernooij et al., 1994).
Moreover, the mechanisms by which SA is transported across the membranes of cells, and/or how this transport may be modulated during defence responses are poorly understood. Studies using tobacco suspension cultures suggest a rapid and transient uptake of SA in a pH-dependent manner, with a linear decrease in uptake between pH 3.5 and pH 8.5 (Chen and Kuc, 1999; Chen et al., 2001). In the roots of Vicia faba and Fagopyrum esculentum SA uptake was prolonged, consisting of an initial passive uptake before the induction of the active uptake mechanism (Shulz et al., 1993); while, in the fronds of Lemna gibba, SA uptake was reported to be linear (Ben-Tal and Cleland, 1982).
In animal systems SA uptake has been more intensively studied. Evidence exists for the presence of a proton-coupled monocarboxylic acid transporter in Caco-2 cells capable of transporting SA (Takanaga et al., 1994). While SA uptake by the human trophoblast cell line was reported to be via a proton-linked active transport system (Emoto et al., 2002). Such import has been linked to a non-specific organic anion transporter (OAT1), which is capable of mediating the transport of a number of anions, including SA, and has been cloned from the mammalian kidney (Sekine et al., 1997).
Evidence is provided here that the uptake of SA by Arabidopsis suspension cultures is driven by proton gradient and the extent to which SA accumulates within the cells is dependent upon the [H+] gradient across the plasmamembrane. Furthermore, using the bacterial elicitor harpin, which has previously been shown to induce a number of defence responses (Baker et al., 1993; He et al., 1994; Desikan et al., 1996, 1998; Dong et al., 1999; Krause and Durner, 2004) similar to those that confer resistance to pathogens (incompatible plant/pathogen interactions, Dangl et al., 1996), it is shown that media alkalization induced by harpin is sufficient to modulate the uptake of SA.
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Materials and methods |
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Arabidopsis suspension cultures and treatments
Cell suspension cultures of Arabidopsis thaliana var. Landsberg erecta were maintained as described by Desikan et al. (1996)
. For uptake experiments, 7-d-old cells were washed twice by centrifugation at 50x g for 5 min in fresh AT3 media (consisting of Murashige and Skoog medium supplemented with 2.5% (w/v) sucrose, 0.5 mg l–1 naphthalene acetic acid, and 0.05 mg l–1 kinetin, pH 5.5), or in assay buffer (consisting of 2.5% (w/v) sucrose, 0.5 mM CaCl2, 0.5 mM K2SO4, and 50 mM of the appropriate buffer: citric acid-sodium citrate buffer pH 5.5–6,2; di-sodium hydrogen orthophosphate-sodium di-hydrogen orthophosphate pH 6.2–8.0), and resuspended at a density of 0.1 g wet wt ml–1. Cells were supplied with 1 µM SA sodium salt (Sigma-Aldrich, UK) supplemented with 20 kBq ml–1 art-461 [ring-3H]-salicylic acid (specific activity 1.85 TBq mmol–1, American Radiolabeled Chemicals Inc, USA). To determine the effects of external pH on the export of SA, cells were loaded in assay buffer at pH 5.5 with 1 µM labelled SA for 30 min, washed and resuspended in the assay buffer at the appropriate pH containing 1 µM non-labelled SA. Cells were treated with catalase (Sigma-Aldrich, UK), superoxide dismutase (SOD, Sigma-Aldrich, UK), sodium nitroprusside (SNP, Sigma-Aldrich, UK), glucose oxidase (Sigma-Aldrich, UK), and nigericin (Molecular Probes, UK) at the indicated concentrations. Harpin was isolated from the Escherichia coli clone pSYH5 containing the DNA sequence encoding harpinPss as described by He et al. (1994) and applied at 1.25 µg ml–1.
Liquid scintillation counting
Estimates of SA uptake by Arabidopsis cells were made by liquid scintillation counting using a Packard tri-carb 2500TR liquid scintillation counter. Cells from 250 µl aliquots of cell suspension were collected by vacuum filtration, washed with 5 ml of the appropriate buffer and dispensed into 2 ml of liquid scintillation fluid (OptiPhase ‘HiSafe’3, Perkin Elmer, UK). The proportion of SA associated with the cells was determined by counting 500 µl of the subsequent wash solution. Non-specific binding was determined as the percentage of SA associate with cellular debris following a freeze–thaw cycle in liquid nitrogen, prior to, or after loading with labelled SA.
HPLC analysis
HPLC analysis was conducted to determine if SA was exported in its native form or as a conjugate. Arabidopsis suspension cultures were set up at 0.2 g wet wt ml–1 in AT3 and treated with 21 µM [7-14C]-salicylic acid (44.44 kBq ml–1, specific activity 2.1 GBq mmol–1, Perkin Elmer Life Sciences, USA). The media from 250 µl aliquots of cell suspension was collected by vacuum filtration at various time points over a 16 h period and stored at –20 °C. HPLC analysis was conducted as described by Bi et al. (1995) using 1.26 kBq of the labelled SA in a final volume of 150 µl.
pH measurements
Measurements of the cytosolic pH of Arabidopsis cells were made using the pH-sensitive ratiometric dye, carboxy seminaphthorhodafluor acetoxymethyl easter acetate (SNARF-1, Molecular Probes, UK) in conjunction with confocal laser scanning microscopy. Cells were treated with SA and maintained on a rotary shaker at room temperature, aliquots were removed and loaded with 5 µM SNARF-1 20 min prior to imaging. Confocal microscopy was performed with the Bio-Rad MRC 1204ES confocal laser scanning microscope controlled with the Bio-Rad Lasersharp 2000 (version 4.1) software interfaced with an inverted Zeiss Axiovert 135 microscope, using an excitation wavelength of 488 nm and dual emission wavelengths of 580 nm and 680 nm as described by Parton et al. (1997). Calibration curves were generated by incubating cells in the presences of 100 mM KCl, 50 mM MES/HEPES, pH 6.2–8.0 adjusted with KOH/HCl and 25 µM nigericin for 60 min prior to imaging (giving a linear relationship between pH 6.2 and pH 8.00, r2=0.98). Changes in the media pH were monitored using a glass pH electrode.
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Results |
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Salicylic acid is rapidly taken up by Arabidopsis suspension cultures in a pH-dependent manner
To determine the kinetics of SA uptake by Arabidopsis suspension cultures, cells were treated with 1 µM SA supplemented with 20 kBq 3H-SA ml–1 (specific activity 1.85 TBq mmol–1) in buffered assay media. Figure 1a illustrates that SA was rapidly taken up by Arabidopsis suspension cultures, with 44.2±2.2%, 28.5±0.9%, and 12.1±1.0% of the applied radioactivity associated with the cells within 2 h of application at pH 5.5, pH 5.9, and pH 6.3, respectively. Since the density of the cells was approximately 12.5% (v/v), the uptake at pH 5.5 represents an accumulation of SA within the cells of 5–6 times that of the surrounding media. Following the initial accumulation of SA, the radioactivity associated with the cells gradually declined at pH 5.5, falling to c. 31.1±3.4% within 6 h, while at pH 6.3, the SA associated with the cells appeared to reach an equilibrium approximately equal to the volume of the cells, with little or no reduction over time (Fig. 1a). The radioactivity associated with cellular debris following a freeze–thaw cycle in liquid nitrogen before or after the addition of SA was minimal (<0.5%) suggesting that SA was taken up by the cells and not simply bound to their surface or cell wall (data not shown). Due to the apparent differences in SA uptake at pH 5.5 and pH 6.3 by Arabidopsis cells, the initial uptake of SA was investigated in assay buffer, buffered between pH 5.5 and pH 6.5. The radioactivity associated with the cells 30 min after the addition of SA declined sharply between pH 5.7 (28.6±2.6%) and pH 6.1 (9.8±0.3%, Fig. 1b), suggesting the uptake of SA is exquisitely pH-dependent within a narrow range. Subsequently, cells were allowed to accumulate SA at pH 5.5 for 30 min (average loading 30.3±0.7% of the applied radioactivity), then transferred to the assay buffer, buffered between pH 5.5 and pH 6.5, to investigate the effects of medium pH on SA export. The radioactivity associated with the cells at 4 h decreased with increasing external pH (Fig. 1c), falling from 28% to 23% at pH 5.5 and from 29% to 9% at pH 6.3 (Fig. 1c). These data suggest that the export of SA is also pH-dependent, however, since the import of SA is pH-dependent (Fig. 1b), export may occur independently of media pH, but against a continued pH-dependent import, with a higher external pH inhibiting import and favouring a greater net export. To determine if the import of SA is a continuous process, cells were incubated in assay buffer at pH 5.5 or pH 6.3 in the presence of unlabelled SA for 2 h, then transferred to fresh assay buffer at pH 5.5 and pH 6.3 containing labelled SA, and the radioactivity associated with the cells was estimated at 30 min. The radioactivity associated with the cells loaded with unlabelled SA at pH 5.5 or pH 6.3, then transferred to fresh assay buffer at pH 5.5 for 30 min approximated that of cells treated with labelled SA for 2.5 h at pH 5.5. Whereas, the radioactivity associated with the cells transferred from unlabelled media at pH 5.5 to labelled media at pH 6.3 was reduced and approximated that associated with cells loaded with SA for 2.5 h at pH 6.3 (Fig. 1d). These data suggest that the import of SA is a continuous process and occurs concurrently with export and that the effects of media pH on the net export of SA result from an inhibition of import.
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In tobacco suspension cultures, SA was reported to be exported without conjugation as free SA (Chen et al., 2001
), HPLC analysis conducted on media and cell extracts collected from Arabidopsis cells challenged with 14C-SA (21 µM, specific activity 2.1 GBq mmol–1), all eluted as a single peak at c. 19 min, corresponding to the SA standards (data not shown), indicating that, in Arabidopsis cell suspension cultures, SA was also exported as free SA with no evidence of conjugation.
The import of salicylic acid requires a proton gradient
The addition of 1 µM SA to Arabidopsis suspension cultures induced a small, but gradual increase in the pH of the media of c. 0.21±0.05 pH units over the controls within 6 h (Fig. 2a). At higher concentrations (1 mM), SA induced a rapid increase in media alkalization, with an initial rate of change of 0.184±0.02 pH units min–1, raising the pH of the media by 0.7±0.13 pH unit over controls within 2 h (Fig. 2a). The addition of SA (1 mM) to the media alone had no measurable effect on pH (data not shown). The rapid changes in media pH suggested that SA import may linked with that of protons, and therefore the effect of SA on the cytosolic pH of the cells was investigated using the pH-sensitive dye SNARF-1. Changes in the cytosolic pH were barely detectable when cells were treated with 1 µM SA, possibly reflecting the small increase in extracellular pH. However, the addition of 1 mM SA induced a rapid and transient decrease in cytosolic pH from 6.83±0.33 to 6.26±0.18 within 1 h, before a recovery to pH 7.07±0.18 within 2 h (Fig. 2b). In addition, the uptake of SA was reduced to less than 5% in cells pretreated for 30 min with the ionophore nigericin (25 µM), which equilibrates cytosolic and extracellular pH by abolishing H+ gradients across membranes (Fig. 2c). The cytoplasmic acidification, media alkalization in response to SA, and the effects of nigericin treatments suggest that SA uptake is driven by a proton gradient.
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Harpin induces media alkalization which inhibits SA uptake
An early event during incompatible plant/pathogen interactions is the alkalization of the plant cell wall, which coincides with the generation of key defence signal ROS and NO (Dangl et al., 1996
; Neill et al., 2002). Given the sharp decline in SA uptake within a narrow pH range, the elicitor harpin was used to investigate how SA transport may be modulated during early defence responses. The bacterial elicitor harpin has previously been shown to elicit ROS (Desikan et al., 1996) and NO (Krause and Durner, 2004) production in Arabidopsis, and media alkalization in tobacco (Baker et al., 1993). Additions of harpin (1.25 µg ml–1) to Arabidopsis cells induced a rapid and sustained alkalization of the cell suspension media, with an initial increase of 0.7±0.2 pH units over the controls within 60 min (Fig. 3a), raising the pH of the media to c. pH 5.97±0.05.
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Co-treatment of cells with harpin (1.25 µg ml–1) and SA (1 µM) had no effect on the kinetics of SA uptake over the initial 60 min period, with 42.01±0.76% and 40.1±0.39% of the applied SA associated with the cells treated with SA alone and SA and harpin at 60 min, respectively (Fig. 3b). However, the radioactivity associated with the cells treated with harpin was reduced at later time points with 24.45±1.81% of the applied radioactivity associated with the cells co-treated with SA and harpin compared with 29.57±1.06 treated with SA alone at 4 h (Fig. 3b). Pretreatment with harpin significantly reduced the uptake of SA over the initial 30 min period reducing the radioactivity associated with the cells by 82% of the controls (SA alone) when cells were pretreated with harpin for 4 h (Fig. 3c). Such inhibition of SA uptake following pretreatment with harpin was abolished in buffered assay media at pH 5.5 (Fig. 3d). Evan's blue staining revealed no loss in cell viability 4 h after harpin treatments, suggesting that the reduction in SA uptake was not due to an increase in cell death. No increase in SA production was found following HPLC analysis of cellular and media extracts obtained at various time points over a 6 h period following the challenge with harpin (1.25 µg ml–1), suggesting that the inhibition of SA uptake was not a result of increased levels of endogenous SA (data not shown).
Pretreatment with SOD (100 U ml–1), or catalase (100 U ml–1) prior to the addition of SA and harpin had little effect on the kinetics of SA import or export compared with cells treated with SA and harpin alone. Similarly, the addition of glucose/glucose oxidase (25 mM/0.01 U ml–1), a concentration which generates H2O2 (20 µM) in the presence of cells at comparable levels to those induced by harpin had no effect on the import/export of SA (Table 1). In addition, pretreatment with the NO donor SNP (0.5 mM) had no effect on the kinetic of SA import/export (Table 1). These data suggest that, of the early defence responses induced by harpin, it is media alkalization that inhibits the accumulation of SA by Arabidopsis cells and facilitates its net export into the media.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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It has been reported here that SA is rapidly taken up by Arabidopsis cells with similar kinetics to those reported for tobacco (Chen and Kuc, 1999
; Chen et al., 2001) and soybean (Dean et al., 2003) when applied in assay buffer at pH 5.5, a pH which mimics that of the growth media of the cells. In unbuffered media the uptake of SA by Arabidopsis cells was associated with media alkalization and cytosolic acidification, suggesting that SA import is linked to that of protons. Such import was reported to be pH-dependent in tobacco, with uptake inversely correlated to increasing media pH between pH 3.5 and pH 8.5 (Chen and Kuc, 1999). In Arabidopsis, using buffered assay media within the physiological pH range of the cell suspension cultures (pH 5.5–6.5), SA uptake was also found to be pH-dependent, but, declined sharply between pH 5.7 and pH 6.1. Such a decline in SA uptake corresponds with a 2.5-fold reduction in the [H+] of the assay buffer, representing a decrease in the [H+] from 1.99 µmol l–1 at pH 5.7 to 0.79 µmol l–1 at pH 6.1, and suggests a requirement for a proton gradient for SA uptake. Pretreating Arabidopsis cells with nigericin, which equilibrates the cytosolic and extracellular pH, abolishing proton gradient across the plasma membrane, severely reduced SA uptake. Together, these data provide evidence that the uptake of SA is linked with that of hydrogen ions and is driven by an inward proton gradient, suggesting mediated transport. A similar requirement for a proton gradient for SA uptake has been reported in human trophoblast cell lines, where import was mediated by a proton-linked active transport system (Emoto et al., 2002). Interestingly, in Arabidopsis the pH of the cytoplasm recovered within 2 h, whereas the pH of the media did not, suggesting that the imported H+ are sequestered into the vacuole or organelles following import.
In tobacco suspension cultures treated with comparable levels of SA, Chen et al. (2001) reported that 50% of the imported SA was exported into the media as free SA within 5 h of application. In Arabidopsis suspension cultures, SA was also exported as free SA with no evidence of conjugation. However, in Arabidopsis, the import and export of SA occurs concurrently and the extent to which SA accumulates within the cells is dependent upon the [H+] of the media. That is, in cells loaded and maintained at pH 5.5 the SA associated with the cells was reduced by 30% within 6 h, whereas cells loaded at pH 5.5 and moved to pH 6.3, the SA associated with them was reduced by 72% within 4 h and approximated that associated with cells loaded and maintained at pH 6.3 for 4 h. In addition, pretreating cells with unlabelled SA prior to the addition of labelled SA suggest that SA import was a continuous process, and that the reduction in the SA associated with cells loaded at pH 5.5 and moved to pH 6.3 was due to an inhibition of SA uptake leading to a net export of SA. Clearly the SA associated with the cells at pH 5.5 was reduced over time, suggesting, either: the up-regulation of an active export mechanism; or the down-regulation of the import mechanism in conjunction with passive or mediated diffusion, or both. In tobacco, Chen et al. (2001) reported both Ca2+-dependent and Ca2+-independent SA export mechanisms functioning at different concentrations of exogenously applied SA. These kinetics of SA transport differ from those reported in planta in which SA uptake is prolonged, resulting in conjugation (Shulz et al., 1993; Ben-Tal and Cleland, 1982), and may reflect the different ways in which the potential phytotoxic effects of exogenously applied SA are ameliorated.
Since the extent to which SA accumulates within Arabidopsis cells is dependent upon the pH of the media and the magnitude of the proton gradient across the plasma membrane, and an early response during incompatible plant/pathogen interactions is the alkalization of the plant cell wall (Dangl et al., 1996), the elicitor harpin was used to investigate how SA uptake may be modulated during defence responses. Harpin has previously been shown to induce defence responses in plants and tissue cultures, including media alkalization in tobacco (Baker et al., 1993), the generation of ROS (Desikan et al., 1996) and NO (Krause and Durner, 2004), and the induction of PCD (Desikan et al., 1998) and SAR in Arabidopsis (Dong et al., 1999). It is report here that harpin induced a rapid and sustained alkalization of the culture media of Arabidopsis cells with similar kinetics to that previously reported in tobacco (Baker et al., 1993). Such alkalization has been linked with a K+/H+ exchange in tobacco (Hoyos et al., 1996) and the induction of an outward rectifying K+ channel in Arabidopsis (El-Maarouf et al., 2001).
In Arabidopsis, the media alkalization induced by harpin reached the critical pH (pH 5.9–6.1), at which the uptake and accumulation of SA is inhibited, at c. 60 min, and coincided with the onset of a reduction in the SA associated with the cells co-treated with harpin at later time points. Whereas pretreatment with harpin for 60 min led to a 54% reduction in SA uptake by Arabidopsis cells. In vitro studies have shown that harpin forms ion-conducting pores in lipid membranes permeable to cations and may also be responsible for nutrient leakage during infection (Lee et al., 2001). However, the formation of such pores is unlikely to be mediating the transmembrane transport of SA since the inhibition of SA uptake induced by harpin was abolished in media buffered to maintain a constant proton gradient. A potential reason for the inhibition of SA uptake in cells treated with harpin is the accumulation of endogenous SA. Samuel et al. (2005) have recently demonstrated that SA accumulates in tobacco leaves infiltrated with harpin within 24 h, whereas, harpin-induced SAR was abolished in nahG tobacco plants which are unable to accumulate SA (Dong et al., 1999). However, no evidence of increased levels of endogenous SA were found in cells treated with harpin over a 6 h period. Together, these data suggest that the media alkalization induced by harpin is sufficient to modulate the kinetics of SA transport by Arabidopsis cells.
In addition to media alkalization, key early events during incompatible plant/pathogen interactions are the generation of ROS and NO which act as signalling molecules for the induction of plant defences and the initiation of PCD (Neill et al., 2002). Salicylic acid has been linked to the enhanced generation of ROS and ensuing cell death in response to avirulent bacteria (Shirasu et al., 1997) and more recently NO has been shown to augment SA induced-SAR in tobacco (Song and Goodmann, 2001). However, pretreating cells with scavengers of ROS or compounds which generate H2O2 or NO had little effect on the kinetics of SA import or export, suggesting that neither modulate SA transport in Arabidopsis and that this is unlikely to be a mechanism by which these compounds enhance SA-dependent events.
Using Arabidopsis suspension cultures as a model system, the kinetics of SA transport have been investigated and how these may be modulated during plant defence responses. These data provide evidence that the import of SA occurs concurrently with export and the extent to which SA accumulates within Arabidopsis cells is dependent upon the magnitude of the proton gradient across the plasma membrane which drives the import mechanism. Furthermore, media alkalization induced by harpin is sufficient to disrupt the proton gradient required for the import of SA, inhibiting uptake and promoting a net export. With this in mind, a model is proposed in which extracellular alkalization induced during incompatible plant/pathogen interaction could modulate the distribution of SA within the plant tissue surrounding the site of attempted infection. Such alkalization would promote the net export of SA to the apoplast at sites of immediate pathogen ingress, which in turn could diffuse to uninfected tissue with a lower apoplastic pH and accumulate within the cells to enhance defence responses. While the kinetics of harpin-induced media alkalization differ from those reported following challenge with avirulent bacteria (Baker et al., 1993), crucially, alkalization and ROS production occur concurrently in both systems. Thus, the uptake of SA would be unaffected during the initial phase of infection, and still able to augment ROS, enhancing defence responses and the initiation of PCD (Mur et al., 1996; Shirasu et al., 1997; Samuel et al., 2005). In Arabidopsis suspension cultures, a 60 min exposure to H2O2 is sufficient to initiate an irreversible commitment to cell death (Desikan et al., 1998), therefore the inhibition of SA uptake and net export of SA would occur after the induction of defence responses. While the nature of SA exported may differ in tissue culture to that reported in planta, free SA in the former (this study, Chen and Kuc, 1999; Chen et al., 2001) and SA ß-glucoside (SAG) in the latter (Hennig et al., 1993; Seo et al., 1995), SAG is readily hydrolysed to yield free SA by extracellular ß-glucosidases in the intercellular spaces of plants (Seo et al., 1995). The uptake of SA released by ß-glucosidases would be inhibited by apoplastic alkalization, and would be free to diffuse to and accumulate in uninfected tissue in a wave preceding infection.
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Acknowledgements |
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We would like to thank Professor SJ Neill for the Arabidopsis suspension cultures and E. coli clone pSYH5 containing the DNA encoding harpinPss. This research was supported by Leverhulme Trust project grant F/00 424/D.
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Footnotes |
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Abbreviations: NO, nitric oxide; PCD, programmed cell death; ROS, reactive oxygen species; SA, salicylic acid; SAR, systemic acquired resistance; SNARF-1, carboxy seminaphthorhodafluor acetoxymethyl easter acetate; SNP, sodium nitroprusside; SOD, superoxide dismutase.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Baker CJ, Orlandi EW, Mock NM. 1993. Harpin, an elicitor of the hypersensitive response in tobacco caused by Erwinia amylovora, elicits active oxygen production in suspension cells. Plant Physiology 102, 1341–1344.[Abstract]
Ben-Tal Y, Cleland CF. 1982. Uptake and metabolism of [14C]salicylic acid in Lemna gibba G3. Plant Physiology 70, 291–296.
Bi YM, Kenton P, Mur L, Darby R, Draper J. 1995. Hydrogen-peroxide does not function downstream of salicylic-acid in the induction of pr protein expression. The Plant Journal 8, 235–245.[CrossRef][Web of Science][Medline]
Chen HJ, Hou WC, Kuc JJ, Lin YH. 2001. Ca2+-dependent and Ca2+-independent excretion modes of salicylic acid in tobacco cell suspension culture. Journal of Experimental Botany 52, 1219–1226.
Chen HJ, Kuc JJ. 1999. Ca2+-dependent excretion of salicylic acid in tobacco cell suspension cultures. Botanical Bulletin of Academia Sinica 40, 267–273.
Dangl JL, Dietrich RA, Richberg MH. 1996. Death don't have no mercy: cell death programs in plant-microbe interactions. The Plant Cell 8, 1793–1807.[CrossRef][Web of Science][Medline]
Dean JV, Shah RP, Mohammed LA. 2003. Formation and vacuolar localization of salicylic acid glucose conjugates in soybean suspension cultures. Physiologia Plantarum 118, 328–336.[CrossRef]
Desikan R, Hancock JT, Coffey MJ, Niell SJ. 1996. Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme. FEBS Letters 382, 213–217.[CrossRef][Web of Science][Medline]
Desikan R, Reynolds A, Hancock JT, Niell SJ. 1998. Harpin and hydrogen peroxide both initiate programmed cell death but have differential effects on defence gene expression in Arabidopsis suspension cultures. Biochemical Journal 330, 115–120.
Dong H, Delaney TP, Bauer DW, Beer SV. 1999. Harpin induces disease resistance in Arabidopsis through the systemic aquired resistance pathway mediated by salicylic acid and the NIM1 gene. The Plant Journal 20, 207–215.[CrossRef][Web of Science][Medline]
EL-Maarouf H, Barny MA, Rona JP, Bouteau F. 2001. Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells. FEBS Letters 497, 82–84.[CrossRef][Web of Science][Medline]
Emoto A, Ushigome F, Koyabu N, Kajiya H, Okabe K, Satoh S, Tsukimori K, Nakano H, Ohtani H, Sawada Y. 2002. H+-linked transport of salicylic acid, an NSAID, in the human trophoblast cell line BeWo. American Journal of Physiology and Cell Physiology 282, 1064–1075.
Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J. 1993. Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261, 754–756.
He SY, Bauer DW, Collmer A, Beer SV. 1994. Hypersensitive response elicited by Erwinia amylovora harpin requires active plant metabolism. Molecular Plant–Microbe Interactions 7, 289–292.
Hennig J, Malamy J, Grynkiewicz G, Indulski J, Klessig DF. 1993. Interconversion of salicylic acid signal and its glucoside in tobacco. The Plant Journal 4, 593–600.[CrossRef][Web of Science][Medline]
Hoyos ME, Stanley CW, He SY, Pike S, Pue XA, Novacky A. 1996. The interaction of harpinPss with plant cell walls. Molecular Plant–Microbe Interactions 9, 608–616.
Krause M, Durner J. 2004. Harpin inactivates mitochondria in Arabidopsis suspension cells. Molecular Plant–Microbe Interactions 17, 131–139.
Lee J, Klüsener B, Tsiamis G, et al. 2001. HrpZPsph from the plant pathogen Pseudomonas syringae pv. phaseolicola is exported by the type III secretion pathway and forms an ion-conducting pore in vitro. Proceedings of the National Academy of Sciences, USA 98, 289–294.
Malamy J, Carr JP, Klessig DF, Raskin I. 1990. Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science 250, 1002–1004.
Mauch-Mani B, Métraux JP. 1998. Salicylic acid and systemic acquired resistance to pathogen attack. Annals of Botany 82, 535–540.
Métraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, Raschdorf K, Schmid E, Blum W, Inverardi B. 1990. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250, 1004–1006.
M
lders W, Buchala A, Métraux JP. 1996. Transport of salicylic acid in tobacco necrosis virus-infected cucumber plants. Plant Physiology 112, 789–792.
Mur L, Naylor G, Warner S, Sugars JM, White RF, Draper J. 1996. Salicylic acid potentiates defence gene expression in tissue exhibiting acquired resistance to pathogen attack. The Plant Journal 9, 559–571.[CrossRef]
Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT. 2002. Hydrogen peroxide and nitric oxide as signalling molecules in plants. Journal of Experimental Botany 53, 1237–1247.
Parton RM, Fischer S, Malhó R, Papasouliotis O, Jelitto TC, Leonard T, Read ND. 1997. Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells. Journal of Cell Science 110, 1187–1198.[Abstract]
Samuel MA, Hall H, Krzymowska M, Drzewiecka K, Hennig J, Ellis BE. 2005. SIPK signalling controls multiple components of harpin-induced cell death in tobacco. The Plant Journal 42, 406–416.[CrossRef][Web of Science][Medline]
Sekine T, Watanabe N, Hosoyamanda M, Kanai Y, Endou H. 1997. Expression cloning and characterization of a novel multi-specific organic anion transporter. Journal of Biological Chemistry 272, 18526–18529.
Seo S, Ishizuka K, Ohashi Y. 1995. Induction of salicylic acid ß-glucosidase in tobacco leaves by exogenous salicylic acid. Plant Cell Physiology 36, 447–453.
Shirasu K, Nakajima H, Krishnamachari Rajasekhar V, Dixon RA, Lamb C. 1997. Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signal in the activation of defence mechanisms. The Plant Cell 9, 261–270.[Abstract]
Shulz M, Schnabl H, Manthe B, Schweihofen B, Casser I. 1993. Uptake and detoxification of salicylic acid by Vicia faba and Fagopyrum esculentum. Phytochemistry 33, 291–294.[CrossRef]
Song F, Goodmann RM. 2001. Activity of nitric oxide is dependent on, but is partially required for function of, salicylic acid in the signalling pathway in tobacco systemic acquired resistance. Molecular Plant–Microbe Interactions 14, 1458–1462.
Summermatters K, Sticher L, Métraux JP. 1995. Systemic responses in Arabidopsis thaliana infected and challenged with Pseudomonas syringae pv. syringae. Plant Physiology 108, 1379–1385.[Abstract]
Takanaga H, Tamia I, Tsuji A. 1994. pH-dependent and carrier-mediated transport of salicylic acid across Caco-2 cells. Journal of Pharmacy and Pharmacology 46, 567–570.[Web of Science][Medline]
Vernooij B, Friedrich L, Morse A, Reist R, Kolditz Jawhar R, Ward E, Uknes S, Kessmann H, Ryals J. 1994. Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. The Plant Cell 6, 959–965.[Abstract]
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