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Induction of a sub-systemic oxidative burst by elicitor-stimulated local oxidative burst in potato plant tissues: a possible systemic signaling in systemic acquired resistance
Plant Science 138 (1998) 197 – 208
Induction of a sub-systemic oxidative burst by elicitor-stimulated local oxidative burst in potato plant tissues: a possible systemic signaling in systemic acquired resistance Hae-Jun Park, Noriyuki Doke *, Yoshio Miura, Kazuhito Kawakita, Tomoya Noritake, Hajime Komatsubara Research Field of Plant Pathology, Di6ision of Bioresources and Functions, Department of Biological Mechanism and Function Sciences, Graduate School of Bioagricultural Sciences, Nagoya Uni6ersity, Nagoya 646, Japan Received 23 February 1998; received in revised form 24 April 1998; accepted 25 June 1998
Abstract A hyphal wall component (HWC) elicitor derived from Phytophthora infestans has been known to cause a local induction of oxidative burst (OXB) as well as a systemic OXB and systemic acquired resistance (SAR) in potato plants. The systemic induction of OXB was investigated using potato tuber slices and excised compound leaves. The elicitor-induced local OXB which was monitored as luminol-mediated chemiluminescence (CL), initialy appeared as scattered small spots of CL and then further developed CL spots on the whole elicitor-treated surface of slices. The induction treatment of the local OXB on one surface of a thick slices caused an OXB on the other surface (sub-systemic OXB). Treatment with elicitor in the presence of an inhibitor O-2 generating NADPH oxidase, diphenyleneiodonium, inhibited appearance of both local and sub-systemic appearance of CL. The systemic CL was not observed in the presence of the inhibitor. A point treatment with HWC-elicitor in the center of horizontally sliced tuber slices radially induced OXB on the whole surface of slice. The point treatment at the basal and lateral part of vertically sliced tuber slices caused a radial development of CL from the site near the treated point. Local treatment of leaflets of an excised compound leaf with elicitor induced the local OXB as well as the sub-systemic OXB on the other non-treated leaflets. These observations indicated that elicitor treatment may cause not only local but also sub-systemic OXB in tissues of potato plants, suggesting that the local OXB may provide a mobile signal for induction of a systemic OXB responsible for induction of SAR. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Elicitor-induced local oxidative burst; Sub-systemic oxidative burst; Luminol-mediated chemiluminescence; Systemic signaling; Potato tuber tissue; Image analysis of superoxide generating site
* Corresponding author. Tel./Fax: +81-52-789-4029; e-mail: [email protected]. 0168-9452/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 2 9 - 0
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1. Introduction The mechanisms of systemic signaling for induction of systemic acquired resistance (SAR) in plants has been unclear and complicated. Few studies have been conducted especially on mobile systemic signaling although some promising substances such as salicylic acid [1,2] and methyl salicylic acid [3] have been considered as the signal molecules of SAR. There are cases, however, that involvement of such molecules is rather negative in the induction of SAR [4,5]. An unique aspect had been reported in relation to induction of SAR in potato plants [6], wherein involvement of salicylic acid is still obscure [5]. Potato plants with seven developed leaves, with the lower leaves treated with an elicitor that caused local oxidative burst (OXB) [6] and a systemic acquired resistance (SAR) to Phytophthora infestans [8], enhanced O2− generating activity in upper elicitor-non-treated leaves [7]. The systemic enhancement of O2− generating activity appeared from at least 1 – 7 days after elicitor treatment [7]. In addition, net activities of superoxide dismutase (SOD) and peroxidase (POX) were enhanced in upper leaves from 2 and 3 days after elicitor treatment, respectively. In this system, a SAR against Phytophthora infestans was reported to be induced from 1 to at least 2 weeks [8]. Previous reports suggested that there may be some connections in induction of the SAR between the elicitor-stimulated local OXB and the systemic activation of O2− generating activity (namely, systemic OXB) and that there might be some mobile signals for induction of the systemic OXB through stem from leaves to leaves. We investigated this possible hypothesis of systemic signaling for the induction of SAR using potato plant tuber tissues (stem) and compound leaves. Our experiments were done by time-sequential observations of the site and intensity of active oxygen generation in the slice-surface of potato tuber tissues through an imaging of luminol-mediated chemiluminescence (CL) derived from active oxygens. The present paper reports evidence showing a systemic development of the OXB in potato tuber tissues and leaflets of compound leaves distant from the site of the initial
OXB directly stimulated by the elicitor. The systemicaly induced OXB at a short distance, henceforth, referred to sub-systemic OXB [9–11].
2. Materials and methods
2.1. Plant materials Potato tubers cv. Rishiri with R1 gene to P. infestans and high level of true resistance against its incompatible races were used. The harvested tubers were stored at 4°C until used. Compound leaves were excised from 9-week-old Rishiri potato plants grown in the field in Spring.
2.2. Preparation of tuber slices Tuber slices were prepared by slicing horizontally or vertically against each stem axis, producing horizontal slices (1.0 and 3.5 cm thick) and vertical slices (1.0 cm thick), respectively. The sliced surface was immediately rinsed with distilled water and then incubated under a moist condition for 18 h at 20°C in the dark to increase the cell’s potential to response to elicitors as previously reported [12].
2.3. Preparation of elicitor and fungal inoculum As a known elicitor of the local OXB [12,13], hypersensitive cell death [12] and phytoalexin production [14,15] in potato tuber tissues, and SAR [8], HWC-elicitor was prepared from cultured mycelia of P. infestans, race 1.2.3.4 according to the method previously reported [16]. The lyophilized elicitor was dissolved in 10 mM Tris– HCl buffer (pH 7.4) with sonication.
2.4. Elicitor treatment and application of luminol HWC-elicitor solution was applied by spreading with glass stick on the whole surface of tuber slices (about 10 ml/cm2) or by attaching a small glass wool disk (about 25 mm2) which had absorbed the solution (100 ml) on to a part of sliced surface to conduct a point treatment. In excised compound leaves, HWC-elicitor solution was ap-
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plied by spreading on leaflets, the surface of which were brushed with distilled water containing caborundum (600 mesh, 4 mg/ml) and rinsed 60 min before application. A luminol solution (2.5 mM in 10 mM Tris – HCl buffer, pH 7.4) was uniformly applied to the whole area of HWC-elicitor non-treated tissue surface (about 10 ml/cm2) to determine the subsystemic OXB. Local OXB on leaflets was determined by applying HWC-elicitor together with luminol solution on tissue surface. The concentration of the luminol was optimum for the quantitative CL assay and the amount of the luminol solution on the tissue surface was enough for continuous determination of the CL for at least 1 h. The continuous monitoring of CL for more than 1 h was not carried out since luminol treatment itself gradually initiated a CL from 1 h after application except experiment in Fig. 1.
2.5. Determination of oxidati6e burst Activity and site of active oxygen generation on the HWC-elicitor treated or non-treated surface of potato tuber slices were continuously monitored as photons from luminol-mediated chemiluminescence (CL) by a CCD camera (model C2400-47, Hamamatsu Photonics Ltd.) in the dark chamber at 20°C. The CL was imaged on photographs depending on the strength of the CL (total integral photon counts/5 min) ranking dark blue, light blue, yellow, red and white colors in that order by using a ARGUES-50 image processor (Hamamatsu Photonics Ltd.). Quantitative analyses of the CL intensity at each spot area was done by computing the integral number of photons in the definite area and time.
3. Results
3.1. Appearance of local OXB in HWC-elicitor treated potato tuber slices The luminol-mediated CL on HWC-elicitor treated surface of potato tuber slices has been autophotographically observed by evaluating the integral value of photons as silver grains on a
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sensitive X-ray film and was confirmed to be dependent on active oxygen species since catalase or radical scavenger diminished the CL [13]. In the present experiments, the luminol-mediated CL on the HWC-treated whole surface of horizontally sliced slices of potato tuber was time-sequentially imaged and presented on photographs (Fig. 1). The CL was initially visible as scattered small spots within 10 min after treatment with a mixture of HWC-elicitor and luminol (Fig. 1b). They were imaged as light blue spots. The CL spots gradually increased in number, area and strength, showing light blue, yellow, red and white in the order of CL strength (Fig. 1c and d) until at least 45 min after treatment and then gradually decreased. The CL spots appeared at random on the whole slice surface consisting of parenchymatous tissues in the center area, vascular bundle area and cortex tissues at peripheral area. Buffer treated (control) also showed a few CL spots at the late stage observed, but to a lesser extent. The HWC-elicitor-stimulated CL, which had been demonstrated to be due to an O2− -generating NADPH oxidase [19–22], was not observed in the presence of an inhibitor of O2− -generating NADPH oxidase, diphenylene iodonium (DPI) (100 mM) [27], H2O2-catalyzing enzyme, catalase (100 mg/ml) [13] or a radical scavenger, tiron (1 mM) [13] together with luminol (data not shown). The time course changes of CL producing activity in the HWC-elicitor treated slice surface was determined by counting the number of photons generated for 5 min at every 10 min after HWCelicitor treatment (Fig. 1e). The CL generating activity significantly increased from about 10 min, and then further increased showing a sigmoidal curve until the time observed (60 min) (Fig. 1e). The level of CL calculated as integral value of photons monitored by a CCD camera was dependent on the concentrations of HWC-elicitor applied from 0.1 to 1.0 mg/ml and of luminol solution applied from 0.1 to 2.5 mM as determined by an autophotography [13]. The HWCelicitor caused browning of surface of tissue slices with a visible gradient depending on the concentrations tested. Observations of vertical section of tissue slices under a light microscope showed that the cell browning appeared within only one or
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Fig. 1. Time course observations of the local OXB as images of luminol-mediated CL on the HWC-elicitor treated surface of potato tuber slices which were made by horizontal slicing to stem axis. Photons from the CL were integrally incorporated for each 5 min from 5 (a), 10 (b), 15 (c) and 20 (d) min after HWC-elicitor treatment and imaged as colored spots of dark blue, light blue, yellow, yellowish red and white in the order from weak to strong intensity. Upper slice (HWC): treated with HWC-elicitor (1 mg/ml) and luminol (2.5 mM) in 10 mM Tris–HCl buffer (pH 7.4), lower slice (CONT): treated with only luminol in the buffer. The periphery of each slice was indicated by broken line. (e); Time course analyses as luminol-mediated CL of the activity of local OXB on HWC-elicitor treated surface of potato tuber slices following stimulation. Each value is the average of integral number of photons generated in a area of 1 cm2 for 5 min at the designated times after stimulation with HWC-elicitor in three experiments. Bars mean the standard deviation from the mean. Solid column: treated with HWC-elicitor, shaded column: treated with buffer only.
two cell layers from the elicitor-treated surface even at 1–2 days after treatment with HWC-elicitor at 1 mg/ml (data not shown). This meant that the HWC-elicitor may not systemically permeate even to 1 mm depth (10 – 15 cells layers) in tissue slices.
3.2. Appearance of sub-systemic OXB on non-treated part of HWC-elicitor treated potato tuber slices Potato tubers were horizontally sliced to stem axis with 3.5 cm thick from top to bottom sur-
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faces. Each was vertically sliced into two halves and then aged for 18 h to develop hypersensitive reactivity of surface cells to HWC-elicitor [12]. A point treatment with 100 ml HWC-elicitor at 1 mg/ml was made by application on 25 mm2 of parenchymatous tissue of the bottom surface of one half. The luminol solution was applied on the whole top surface. CL on the luminol-applied surface was continuously monitored and imaged as photographs (Fig. 2A). The CL initially appeared as scattered small spots at random from 20 to 30 min after treatment with HWC-elicitor (Fig. 2A, b). The CL spots gradually increased in number and each developed with increase in size and strength until at least 60 min after induction treatment (Fig. 2A, c and d). Control slices (buffer treatment) revealed systemic CL spots to a lesser degree at around 60 min after treatment (Fig. 2A, c and d). The systemic CL on the elicitor non-treated surface of the elicitor-treated slice was not observed when HWC-elicitor was applied together with the inhibitor of local OXB, DPI (100 mM) (Fig. 3A and B), catabolic enzymes of H2O2, catalase (100 mg/ml) or radical scavenger, tiron (1 mM) (data not shown). The systemic CL in tissue slices treated with HWC-elicitor was not observed when it was assayed in the presence of the above inhibitors. Intensity of the systemic appearance of CL also varied to some extent due to the nature of each tuber slice or storage age. Quantitative analyses showed that the systemic CL significantly appeared around 20 min after induction treatment in the case of 3.5 cm thick slices and reached the maximum level at around 60 min (Fig. 2B). The tissue surface where the sub-systemic OXB appeared did not show visible changes like tissue browning caused by direct treatment with HWCelicitor.
3.3. Appearance of sub-systemic OXB in a compound leaf The upper surface of leaflets at the middle position of an excised compound leaf was applied with a mixture of HWC-elicitor or buffer (control) by carborundum brushing to produce elicitor
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Fig. 2. Time course observations of a sub-systemic oxidative burst as images of luminol-mediated CL on the HWCelicitor non-treated surface of potato tuber tissues (3.5 cm thick) which were made by horizontal slicing to stem axis (A). The slice was vertically cut into two halves for stimulation with HWC-elicitor (HWC) or buffer (CONT). HWCelicitor was applied by attaching a glass wool disc (0.25 mm2) absorbing HWC-elicitor or buffer solution on the parenchymatous tissues of the bottom. Each upper surface of the slice was entirely applied with luminol solution (2.5 mM). Photons from CL were incorporated for each 10 min at 10 (a), 20 (b), 40 (c) and 60 (d) min after stimulation and imaged as described in Fig. 1. Upper and lower sets shows two typical results. (B) Time course analyses of (A). Each value is the average of integral number of photons generated in area of 1 cm2 for every 5 min at designated times after elicitor treatment in three experiments. Bars mean the standard deviation from the mean. Solid column: treated with HWC-elicitor, shaded column: treated with buffer only.
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on almost the whole surface of HWC-elicitor treated leaflets (Fig. 4A, a). The CL was also observed on the non-treated leaflets at the basal and top position to a significant extent. Buffertreated leaflets locally and systemically showed CL spots only at a lesser level (Fig. 4A, b). The compound leaf which was not pre-treated with
Fig. 3. Effect of DPI on the HWC-induced sub-systemic OXB in potato tuber. (A) Images of luminol-mediated CL of effect of DPI on induction of the HWC-induced systemic OXB. Bottom surface of each disc was treated with HWC (1.0 mg/ml) in 10 mM Tris – HCl buffer (pH 7.4) (a and d); buffer only (b); HWC solution (1.0 mg/ml) containing DPI (100 mM) (c). On the upper surface of each disc, luminol (2.5 mM) containing DPI (d), not containing DPI was applied (a, b and c). CL on the upper surface was imaged by incorporation for 5 min after 60 min. Thickness of the tissue used was 3.5 cm. (B) Quantitative assay as luminol-mediated CL of (A). Each value is the average of integral number of photons generated in a area of 1 cm2 for 5 min at the designated times after stimulation with HWC-elicitor in three experiments. Bars mean the standard deviation from the mean.
effect, while the other leaflets were applied with buffer plus carborundum brushing. One hour after elicitor treatment, the CL on the surface of each leaflet was monitored from 10 min in the dark after application of luminol solution on the surface of each leaflet since a CL derived from quenching of chlorophyll appeared. CL appeared
Fig. 4. Imaging analyses as a function of luminol-mediated CL of local and sub-systemic oxidative burst in leaflets of the compound leaves of potato plant. (A) Upper surface of two leaflets located at middle position of an excised compound leaf were treated with HWC-elicitor by brushing with carborundum and other leaflets were only brushed with buffer containing carborundum. Upper surface of all leaflets was applied with luminol 1 h after elicitor treatment, kept in the dark for 10 min, and then subjected to imaging by integral counting of photons for 10 min in the dark. White spots in leaflets mean the area showing CL. (a) The middle leaflets treated with elicitor; (b) the middle leaflets treated with buffer; and (c) all leaflet treated with nothing. Outline of each leaflet was drawn by broken line. (B) Quantitative assay of the sub-systemic OXB in HWC-elicitor non-treated leaflets at top and basal parts of a compound leaf, two leaflets at the middle position of which were treated with HWC-elicitor. Experimental systems and methods were the same as for (A). Each value means the average percent of the systemically appeared CL level to the level of locally appeared CL in elicitor-treated leaflets in three experiments. Bars mean the standard deviation from the mean.
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carborundum scarcely showed CL on each leaflets even if the two leaflets at middle position was applied with elicitor solution (Fig. 4A, c). The intensity of the CL level varied from sample to sample. Thus, percent value of the CL in HWC-elicitor non-treated leaflets to that in the elicitortreated leaflets was calculated in each sample for a comparison of the systematically appeared CL level. At 1 h after HWC-elicitor treatment, the levels of CL on the non-treated leaflets at the basal and top parts reached to about 29 and 34%, respectively (Fig. 4B). These observations indicated that HWC-elicitor treatment caused the OXB not only on directly treated leaflets but also on nontreated leaflets to both directions within 1 h.
3.4. Analyses of two-dimensional mo6ement of the sub-systemic OXB in a potato tuber slice A spot treatment with HWC-elicitor solution was done by attaching a small glass wool disk absorbing HWC-elicitor solution in the center of horizontally sliced tuber tissue (long axis: 4 – 5 cm, and short axis: 3–4 cm). The remaining slice surface was covered by spreading with luminol solution only. A time course charge of the development of CL showed an initial appearance of CL as scattered spots at random on the luminol-treated area from about 10 min after HWC-elicitor treatment (Fig. 5, b). The CL spots developed much stronger and larger with incubation time, and spread on almost the whole surface until about 40 min after the spot treatment (Fig. 5c and d). Buffer-treated tissues revealed a few CL spots at around 40 min. This indicated that elicitor treatment at the center of parenchymatous tissues of horizontally sliced tissues developed the sub-systemic OXB to vertical direction. Distinguished difference in the CL appearance was not observed among parenchymatous, vascular bundle and cortex tissues. A spot treatment with HWC-elicitor was done on the edge at the basal part of vertically sliced tuber tissues (10 mm thick, longitudinal stem axis: 8 – 9 cm long) and the luminol solution was uniformly applied on the whole slice-surface (Fig. 6A, a). Time-sequential observations of the CL development showed an initiation of the CL near the HWC-elicitor-treated part from about 10 min after
Fig. 5. Time course imaging of the sub-systemic OXB as the luminol-mediated CL in horizontally sliced tissues (1 cm thick) in the center of which a spot treatment with HWC-elicitor was done. The elicitor non-treated area was applied with luminol solution. Photons from the CL were incorporated for each 5 min at 10 (a), 20 (b), 30 (c) and 40 (d) min after elicitor treatment. White area on the slice surface means the CL generating area. Periphery of each slice was drawn by the broken line.
treatment. The CL area gradually and radialy developed to the direction of the top area of the tuber slice with a gradient of CL intensity from the basal area. At 60 min after treatment, the top edge of CL area reached nearly half of the tuber slices at the elicitor-treated site (Fig. 6A). At the front of CL area, scattered CL spots appeared during the development. No distinguished appearance of the CL was observed among parenchymatous, vascular bundle and cortex tissues on the sliced surface. At late stages, it was observed that buffer treatment caused the CL development near treated site to a lesser extent (Fig. 6A). From repeated experiments, time course changes in the average values of CL were analyzed in each spot (about 0.25 cm2) at definite distance from the HWC-elicitor treated site. The CL level at the nearest spot increased most strongly with a sigmoidal curve with time (Fig.
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Fig. 6. Time course imaging of the sub-systemic OXB on vertically sliced tissues of potato tuber at the edge of the basal part of which a point treatment with HWC-elicitor was done (A). Luminol solution was spread on the whole surface of the slice (long axis: 8 cm, 10 mm thick). Each image was designated by incorporation of photons generated for 5 min from 10 (b), 20 (c), 30 (d), 40 (e) or 60 (f) min after elicitor treatment. Light blue, yellow, yellow red and white color on the slices mean the area of CL generation and its intensity in that order. Periphery of each slice was drawn by broken line. (B) Time course change in the activity of luminol mediated CL by sub-systemic OXB on vertically sliced tuber tissues at the edge of the basal part of which a point treatment with HWC-elicitor was done. Experimental systems and methods were the same as for (A). The counting spots were set at every 2 cm distant from spot 3 just neighboring to the elicitor treated spot. Each value represents the average value of three experiments showing integral number of photons at each spot designated in inlet figures for each 5 min from every 5 min after elicitor treatment.
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6B). The CL at each spot distant by 2 cm from the basal spot also showed similar increasing curve, but at a half level of that of the basal spot area. The CL at each spot distant by 4 cm from the basal spot was much more lower (about 1/10 of CL level at the basal spot area). (Fig. 6B). Within 60 min after HWC-elicitor treatment, the detectable CL reached to at least 4 or 5 cm from the elicitor-treated site. This indicated that local OXB developed a sub-systemic OXB in a tuber stem tissue to a direction from bottom to top with time.
4. Discussion A hypothesis that the OXB in plant cells caused by direct treatment with HWC-elicitor (local OXB) may trigger a sub-systemic OXB at distance from initially activated cells was investigated using potato tuber tissues (stem tissue). The local and sub-systemic OXB were determined by timesequential and two-dimensional imaging analyses of the luminol-mediated CL due to active oxygen species generated on the surface of tissue-slices. Experimental results evidently demonstrated a development of the CL to tissue area distant from the site with the local OXB caused by direct contact with HWC-elicitor (Fig. 1). Elicitor-stimulated CL on plant tissues [13] or suspension-cultured cells [17] have been shown to be predominantly due to H2O2 since concomitant presence of catalase with luminol diminished the CL appearance [13,18]. Additionally, protoplasts without cell wall rapidly activated a SOD-sensitive extra-cellular cytochrome c reducing activity upon reaction with elicitor, suggesting O2− production outside of protoplasts [19]. Elicitor-stimulated O2− generation in plant cells has been proposed to be dependent on an O2− generating NADPH oxidase in plasma membrane [19,20]. The catalase-inhibitory CL was inhibited by a known inhibitor of O2− generating NADPH oxidase, DPI [27]. The systemically appeared CL may be due to an activation of O2− generating NADPH oxidase similar to that in local OXB since it was also inhibited by DPI (Fig. 3).
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A possibility that H2O2 generated during local OXB moves to a distant place through intercellular space of the tuber tissue by diffusion or capillary movement and causes the sub-systemic CL could be ruled out because of the following reasons. The sub-systemic appearance of CL was also inhibited by DPI. If H2O2 systemically moves to the opposite surface at 3.5 cm distance, DPI could not inhibit the appearance of sub-systemic CL. Furthermore, if HWC-elicitor itself systemically reached to a distant place, browned tissue cells could not be restricted in one or two cell layers from HWC elicitor-treated surface. Our preliminary experiments also showed that other inhibitors of the local OXB such as Ca2 + chelator, EGTA, protein kinase inhibitor, staurosporine etc. inhibited the induction of sub-systemic OXB [10]. The system for the subsystemic OXB could be similar to that for the local OXB. Why and by what the sub-systemic OXB is activated remains an interesting subject in relation to systemic signaling in plant tissues. The local and sub-systemic OXB were interestingly characterized by initial appearance of scattered spots in tissues (Fig. 1A and 2A). The CL area developed further by enlarging of each CL spot and increasing the spot number to the whole tissue surface. It seemed that some characteristic cells in the tissues may sensitively recognize the elicitor or initiate the active oxygen generating reaction following treatment with HWC-elicitor. In the case of sub-systemic OXB, however, some cells may be assumed to accept a systemic signal from the original cells with OXB other than elicitor molecule itself. The initial CL spots in sub-systemic OXB became like cores for further development of the CL to neighboring cells through cell to cell. This assumption raises a possibility that specific cells responsible for the initial OXB may be differentiated in the potato tuber tissues and that there may be some systemic signals which could activate the OXB from cell to cell. It is also interesting to consider the previous observations that potato tuber tissues provided both protoplasts with and without hypersensitive reactivity to HWC-elicitor [16]. What signal system may be involved in the induction of sub-systemic OXB is obscure in the
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present experiment. The data showed that inhibition of the induction of the local OXB by DPI (Fig. 3A and 3B) or scavengers of generated active oxygen, catalase or tiron resulted in little appearance of the sub-systemic OXB. So, we assumed that the sub-systemic OXB could be triggered by unknown mobile signal system stimulated by active oxygen species generated during local OXB. The present experiments showed that inhibition of the local CL in the presence of catalase resulted in less appearance of the sub-systemic CL. These preliminary results suggested that the sub-systemic CL may be triggered mainly by H2O2 generated during the local OXB, which is dismutated from O2− generated by the NADPH oxidase. Our preliminary experiments also showed that exogeneously applied H2O2 solution caused an induction of sub-systemic CL in potato tuber tissues [10]. Therefore, H2O2 produced in the local OXB could be a potential trigger of the sub-systemic OXB. The mechanisms of development of the OXB from cell to cell remain to be clarified in relation to the systemic signaling for the response against elicitor in plant tissues. The present experiment using excised compound leaves showed a possibility of the systemic OXB between leaflets in a compound leaf (Fig. 4A and 4B). Application of a mixture of HWC-elicitor and luminol solutions on the surface of leaflets of excised compound leaves caused a CL, indicating the induction of local OXB in leaf tissues (Fig. 4A, a). A treatment of two leaflets at middle position of a compound leaf with HWC-elicitor also showed a sub-systemic CL on the other leaflets treated only with luminol (Fig. 4A, b). This suggested an appearance of sub-systemic OXB in excised compound leaf. The present observations of local and sub-systemic OXB among the leaflets of excised compound leaves may be connected to induction of a possible systemic OXB in whole plants as shown previously [23] through petiole from leaflets and the main stem from compound leaves. The time-sequential observation of sub-systemic OXB in vertically sliced tuber tissues (Fig. 6B) suggested a possibility of subsequent development of OXB from cell to cell in stem tissues. It is assumed that some signals for sub-systemic OXB
may move through the main stem of potato plant. Since the present experiments allowed a continuous observation of CL for only 1 h after elicitorstimulation, movement of the OXB to the distant part was confirmed by no more than 5 cm during observation. It is necessary to devise some methods for determination of OXB for longer periods and distance in the whole plant. Application of HWC-elicitor and luminol solutions on leaflets was done by brushing with carborundum since there is little response of epidermal cells to these agents without a wound. Without carborundum-wounding, little CL was observed even in leaf tissues applied with HWCelicitor. The cuticle layer of leaflets seems to reject HWC-elicitor or luminol to the reaction site in tissue cells. The observation of CL in the leaflet was done for a limited time from 10 min after starting the CL observation in the dark. It was because plant tissues containing chlorophyll reflect a CL for several minutes immediately after moving from light to dark conditions. The CL due to the local OXB in leaflets tended to decline with increase in incubation time for several minutes in the dark. The decline of the elicitor-stimulated CL can not be recovered in the dark since NADPH generated during photosynthesis could be possibly associated with the OXB. The elicitorstimulated OXB in potato plant leaflets had been detected as SOD-sensitive reduction of cytochrome c in the light [7]. As potato tuber tissues provide NADPH through activated pentose mono-phoshate shunt in the course of resistance response [24], the CL seems to continue for a long time in the dark. In the present experimental systems, we did not determine the induction of SAR in relation to induction of sub-systemic OXB since potato tuber (storage stem) slices and detached compound leaves were not appropriate for determination of SAR. Local treatment of leaves of potato plants with HWC-elicitor had been shown to induce a systemic OXB [7] as well as SAR against P. infestans [8]. Furthermore, potato plants grown from potato tuber treated with HWC-elicitor had been shown to acquire a SAR [25,26]. In association with these reports, it is assumed that local OXB could trigger the systemic OXB and SAR in
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potato plants, in which an inducer of SAR did not induce systemic synthesis of salicylic acid [5]. The present paper only demonstrated an induction of novel sub-systemic OXB by inducer of both local OXB and SAR in restricted tissues of potato plants.
Acknowledgements The authors appreciate Drs H. Yoshioka and T. Tsuge for helpful discussion and Y. Umemura for supplying potato tuber seeds. The first author acknowledges the scholarship grants from Yoneyama Rotary Foundation (1994 – 1996) and the Ministry of Education, Science and Culture of Japan (1996–1998). This work was partly supported by grant-in-aid for Exploratory Research (No. 09876092) and for Scientific Research (B) (No. 09460027) from the Ministry of Education, Science and Culture.
References [1] J. Malamy, P. Carr, D.F. Klessig, I. Raskin, Salicylic acid: A likely endogenous signal in the resistance response of tobacco to viral infection, Science 250 (1990) 1002– 1004. [2] W. Mo¨lder, A. Buchala, J.P. Me´traux, Transport of salicylic acid in tobacco necrosis virus-infected cucumber plants, Plant Physiol. 112 (1996) 787–792. [3] V. Shulaev, P. Silverman, L. Raskin, Airborne signaling by methyl salicylate in plant pathogen resistance, Nature 385 (1997) 718 – 721. [4] B. Vernooij, L. Friedrich, A. Morse, et al., Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction, Plant Cell 6 (1994) 959–965. [5] J.-L. Coquoz, A.J. Buchala, Ph. Meuwly, J.-P. Me´traux, Arachidonic acid induces local but not systemic synthesis of salicylic acid and confers systemic resistance in potato plants to Phytophthora infastans and Alternaria solani, Phytopathology 85 (1995) 1219–1224. [6] H.B. Chai, N. Doke, Superoxide anion generation: a response to potato leaves to infection with Phytophthora infestans, Physiol. Biochem. 77 (1987) 645–649. [7] H.B. Chai, N. Doke, Systemic activation of O2− generation reaction, superoxide dismutase, and peroxidase in potato plants to Phytophthora infestans, Ann. Phytopath. Soc. Jpn. 52 (1987) 585–590.
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[8] N. Doke, A.V. Ramirez, K. Tomiyama, Systemic induction of resistance in potato plants against Phytophthora infestans by local treatment with hyphal wall components of the fungus, J. Phytopathol. 119 (1987) 232 – 239. [9] H.-J. Park, N. Doke, Y. Miura, T. Noritake, Systemic signal transduction in induced resistance of plant: systemic as well as local activation of superoxide generating system in potato tuber tissue treated with fungal elicitor, Ann. Phytopath. Soc. Jpn. 60 (1994) 767 Abstract in Japanese. [10] H.-J. Park, N. Doke, Y. Miura, K. Kawakita, T. Noritake, Induction of fungal elicitor-stimulated systemic activation of superoxide generation by inhibitors of signal transduction, Ann. Phytopath. Soc. Jpn. 61 (1995) 245 Abstract in Japanese. [11] H.-J. Park, Y. Miura, T. Noritake, K. Kawakita, N. Doke, Imaging analysis of appearance of local and subsystemic oxidative bursts in elicitor-treated potato tuber slices, Ann. Phytopath. Soc. Jpn. 62 (1996) 291 Abstract in Japanese. [12] N. Doke, Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components, Physiol. Plant Pathol. 23 (1983) 345 – 357. [13] Y. Miura, H. Yoshioka, N Doke, An autophotograpic determination of the active oxygen generation in potato tuber discs during hypersensitive response to fungal infection or elicitor, Plant Sci. 105 (1995) 45 – 52. [14] N. Lisker, J. Kuc, Elicitation of terpenoid accumulation in potato tuber tissue, Phytopathology 67 (1977) 1356 – 1359. [15] S. Sakai, N. Doke, K. Tomiyama, Relation between necrosis and rishitin accumulation in potato tuber slices treated with hyphal wall components of Phytophthora infestans, Ann. Phytopath. Soc. Jpn. 48 (1982) 238 – 240. [16] N. Doke, K. Tomiyama, Effect of hyphal wall components from Phytophthora infestans on protoplasts of potato tuber tissues, Physiol. Plant Pathol. 16 (1980) 169 – 176. [17] L.M Sanchez, N. Doke, K. Kawakita, Elicitor-induced chemiluminescence in cell suspension cultures of tomato, sweet pepper and tobacco plants and its inhibition by suppressors from Phytophthora spp, Plant Sci. 88 (1993) 141 – 148. [18] W.A. Lindner, C. Hoffmann, H. Grisebach, Rapid elicitor-induced chemiluminescence in soybean cell suspension cultures, Phytochemistry 27 (1988) 2501 – 2503. [19] N. Doke, NADPH-dependent O2− generation in membrane fractions isolated from wounded potato tubers inoculated with Phytophthora infestans, Physiol. Plant Pathol. 27 (1985) 311 – 322. [20] N. Doke, Y. Miura, In vitro activation of NADPH-dependent O2− generating system in a plasma menbranerich fraction of potato tuber tissues by treatment with an elicitor from Phytophthora infestans or with digitonin, Physiol. Mol. Plant Pathol. 46 (1995) 17 – 28.
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[21] F. Rossi, The O2− -forming NADPH oxidase of the phagocytes: nature mechanisms of activation and function, Biochem. Biophys. Acta. 853 (1986) 65–89. [22] M.C. Mehdy, Active oxygen species in plant defense against pathogens, Plant Physiol. 105 (1994) 467– 472. [23] H.-J. Park, H. Komatsubara, K. Kawakita, N. Doke, An Imaging analysis of local and subsystemic oxidative bursts in elicitor-treated potato compound leaf and tuber slice, Ann. Phytopath. Soc. Jpn. 62 (1996) 620 (Abstract in Japanese). [24] K. Graeve, A. von Schaewen, R. Scheibe, Purification, characterization, and cDNA sequence of glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.), Plant J. 5 (1994) 353–361.
[25] N. Doke, T. Hikota, H. Kawamura et al., Plant immunization in potato culture: its basis and application, in: E.T. Rasco, Jr., F.B. Aromin, C.H. Balatero (Eds.), Proceedings of the Fourth APA Triennial Conference, 2 (1994) 29 – 36. [26] N. Doke, T. Hikota, H.B. Chai, Y. Miura, K. Kawakita, New approaches for protection of potao Plants against Late Blight: Enhancement of incompatibility by regulation of the mechanism of host – parasite interaction, in: Q. Liu, T. Kokubu (Eds.), Proceedings of the 1st Chinese – Japanese Symposium on Sweetpotato and Potato, Beijing Agricultural University Press, Beijing, 1995, pp. 341 – 346. [27] J. Hancock, O. Jones, The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophage, Biochem J. 242 (1987) 103 – 107.
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Induction of a sub-systemic oxidative burst by elicitor-stimulated local oxidative burst in potato plant tissues: a possible systemic signaling in systemic acquired resistance Hae-Jun Park, Noriyuki Doke *, Yoshio Miura, Kazuhito Kawakita, Tomoya Noritake, Hajime Komatsubara Research Field of Plant Pathology, Di6ision of Bioresources and Functions, Department of Biological Mechanism and Function Sciences, Graduate School of Bioagricultural Sciences, Nagoya Uni6ersity, Nagoya 646, Japan Received 23 February 1998; received in revised form 24 April 1998; accepted 25 June 1998
Abstract A hyphal wall component (HWC) elicitor derived from Phytophthora infestans has been known to cause a local induction of oxidative burst (OXB) as well as a systemic OXB and systemic acquired resistance (SAR) in potato plants. The systemic induction of OXB was investigated using potato tuber slices and excised compound leaves. The elicitor-induced local OXB which was monitored as luminol-mediated chemiluminescence (CL), initialy appeared as scattered small spots of CL and then further developed CL spots on the whole elicitor-treated surface of slices. The induction treatment of the local OXB on one surface of a thick slices caused an OXB on the other surface (sub-systemic OXB). Treatment with elicitor in the presence of an inhibitor O-2 generating NADPH oxidase, diphenyleneiodonium, inhibited appearance of both local and sub-systemic appearance of CL. The systemic CL was not observed in the presence of the inhibitor. A point treatment with HWC-elicitor in the center of horizontally sliced tuber slices radially induced OXB on the whole surface of slice. The point treatment at the basal and lateral part of vertically sliced tuber slices caused a radial development of CL from the site near the treated point. Local treatment of leaflets of an excised compound leaf with elicitor induced the local OXB as well as the sub-systemic OXB on the other non-treated leaflets. These observations indicated that elicitor treatment may cause not only local but also sub-systemic OXB in tissues of potato plants, suggesting that the local OXB may provide a mobile signal for induction of a systemic OXB responsible for induction of SAR. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Elicitor-induced local oxidative burst; Sub-systemic oxidative burst; Luminol-mediated chemiluminescence; Systemic signaling; Potato tuber tissue; Image analysis of superoxide generating site
* Corresponding author. Tel./Fax: +81-52-789-4029; e-mail: [email protected]. 0168-9452/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 2 9 - 0
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1. Introduction The mechanisms of systemic signaling for induction of systemic acquired resistance (SAR) in plants has been unclear and complicated. Few studies have been conducted especially on mobile systemic signaling although some promising substances such as salicylic acid [1,2] and methyl salicylic acid [3] have been considered as the signal molecules of SAR. There are cases, however, that involvement of such molecules is rather negative in the induction of SAR [4,5]. An unique aspect had been reported in relation to induction of SAR in potato plants [6], wherein involvement of salicylic acid is still obscure [5]. Potato plants with seven developed leaves, with the lower leaves treated with an elicitor that caused local oxidative burst (OXB) [6] and a systemic acquired resistance (SAR) to Phytophthora infestans [8], enhanced O2− generating activity in upper elicitor-non-treated leaves [7]. The systemic enhancement of O2− generating activity appeared from at least 1 – 7 days after elicitor treatment [7]. In addition, net activities of superoxide dismutase (SOD) and peroxidase (POX) were enhanced in upper leaves from 2 and 3 days after elicitor treatment, respectively. In this system, a SAR against Phytophthora infestans was reported to be induced from 1 to at least 2 weeks [8]. Previous reports suggested that there may be some connections in induction of the SAR between the elicitor-stimulated local OXB and the systemic activation of O2− generating activity (namely, systemic OXB) and that there might be some mobile signals for induction of the systemic OXB through stem from leaves to leaves. We investigated this possible hypothesis of systemic signaling for the induction of SAR using potato plant tuber tissues (stem) and compound leaves. Our experiments were done by time-sequential observations of the site and intensity of active oxygen generation in the slice-surface of potato tuber tissues through an imaging of luminol-mediated chemiluminescence (CL) derived from active oxygens. The present paper reports evidence showing a systemic development of the OXB in potato tuber tissues and leaflets of compound leaves distant from the site of the initial
OXB directly stimulated by the elicitor. The systemicaly induced OXB at a short distance, henceforth, referred to sub-systemic OXB [9–11].
2. Materials and methods
2.1. Plant materials Potato tubers cv. Rishiri with R1 gene to P. infestans and high level of true resistance against its incompatible races were used. The harvested tubers were stored at 4°C until used. Compound leaves were excised from 9-week-old Rishiri potato plants grown in the field in Spring.
2.2. Preparation of tuber slices Tuber slices were prepared by slicing horizontally or vertically against each stem axis, producing horizontal slices (1.0 and 3.5 cm thick) and vertical slices (1.0 cm thick), respectively. The sliced surface was immediately rinsed with distilled water and then incubated under a moist condition for 18 h at 20°C in the dark to increase the cell’s potential to response to elicitors as previously reported [12].
2.3. Preparation of elicitor and fungal inoculum As a known elicitor of the local OXB [12,13], hypersensitive cell death [12] and phytoalexin production [14,15] in potato tuber tissues, and SAR [8], HWC-elicitor was prepared from cultured mycelia of P. infestans, race 1.2.3.4 according to the method previously reported [16]. The lyophilized elicitor was dissolved in 10 mM Tris– HCl buffer (pH 7.4) with sonication.
2.4. Elicitor treatment and application of luminol HWC-elicitor solution was applied by spreading with glass stick on the whole surface of tuber slices (about 10 ml/cm2) or by attaching a small glass wool disk (about 25 mm2) which had absorbed the solution (100 ml) on to a part of sliced surface to conduct a point treatment. In excised compound leaves, HWC-elicitor solution was ap-
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plied by spreading on leaflets, the surface of which were brushed with distilled water containing caborundum (600 mesh, 4 mg/ml) and rinsed 60 min before application. A luminol solution (2.5 mM in 10 mM Tris – HCl buffer, pH 7.4) was uniformly applied to the whole area of HWC-elicitor non-treated tissue surface (about 10 ml/cm2) to determine the subsystemic OXB. Local OXB on leaflets was determined by applying HWC-elicitor together with luminol solution on tissue surface. The concentration of the luminol was optimum for the quantitative CL assay and the amount of the luminol solution on the tissue surface was enough for continuous determination of the CL for at least 1 h. The continuous monitoring of CL for more than 1 h was not carried out since luminol treatment itself gradually initiated a CL from 1 h after application except experiment in Fig. 1.
2.5. Determination of oxidati6e burst Activity and site of active oxygen generation on the HWC-elicitor treated or non-treated surface of potato tuber slices were continuously monitored as photons from luminol-mediated chemiluminescence (CL) by a CCD camera (model C2400-47, Hamamatsu Photonics Ltd.) in the dark chamber at 20°C. The CL was imaged on photographs depending on the strength of the CL (total integral photon counts/5 min) ranking dark blue, light blue, yellow, red and white colors in that order by using a ARGUES-50 image processor (Hamamatsu Photonics Ltd.). Quantitative analyses of the CL intensity at each spot area was done by computing the integral number of photons in the definite area and time.
3. Results
3.1. Appearance of local OXB in HWC-elicitor treated potato tuber slices The luminol-mediated CL on HWC-elicitor treated surface of potato tuber slices has been autophotographically observed by evaluating the integral value of photons as silver grains on a
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sensitive X-ray film and was confirmed to be dependent on active oxygen species since catalase or radical scavenger diminished the CL [13]. In the present experiments, the luminol-mediated CL on the HWC-treated whole surface of horizontally sliced slices of potato tuber was time-sequentially imaged and presented on photographs (Fig. 1). The CL was initially visible as scattered small spots within 10 min after treatment with a mixture of HWC-elicitor and luminol (Fig. 1b). They were imaged as light blue spots. The CL spots gradually increased in number, area and strength, showing light blue, yellow, red and white in the order of CL strength (Fig. 1c and d) until at least 45 min after treatment and then gradually decreased. The CL spots appeared at random on the whole slice surface consisting of parenchymatous tissues in the center area, vascular bundle area and cortex tissues at peripheral area. Buffer treated (control) also showed a few CL spots at the late stage observed, but to a lesser extent. The HWC-elicitor-stimulated CL, which had been demonstrated to be due to an O2− -generating NADPH oxidase [19–22], was not observed in the presence of an inhibitor of O2− -generating NADPH oxidase, diphenylene iodonium (DPI) (100 mM) [27], H2O2-catalyzing enzyme, catalase (100 mg/ml) [13] or a radical scavenger, tiron (1 mM) [13] together with luminol (data not shown). The time course changes of CL producing activity in the HWC-elicitor treated slice surface was determined by counting the number of photons generated for 5 min at every 10 min after HWCelicitor treatment (Fig. 1e). The CL generating activity significantly increased from about 10 min, and then further increased showing a sigmoidal curve until the time observed (60 min) (Fig. 1e). The level of CL calculated as integral value of photons monitored by a CCD camera was dependent on the concentrations of HWC-elicitor applied from 0.1 to 1.0 mg/ml and of luminol solution applied from 0.1 to 2.5 mM as determined by an autophotography [13]. The HWCelicitor caused browning of surface of tissue slices with a visible gradient depending on the concentrations tested. Observations of vertical section of tissue slices under a light microscope showed that the cell browning appeared within only one or
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Fig. 1. Time course observations of the local OXB as images of luminol-mediated CL on the HWC-elicitor treated surface of potato tuber slices which were made by horizontal slicing to stem axis. Photons from the CL were integrally incorporated for each 5 min from 5 (a), 10 (b), 15 (c) and 20 (d) min after HWC-elicitor treatment and imaged as colored spots of dark blue, light blue, yellow, yellowish red and white in the order from weak to strong intensity. Upper slice (HWC): treated with HWC-elicitor (1 mg/ml) and luminol (2.5 mM) in 10 mM Tris–HCl buffer (pH 7.4), lower slice (CONT): treated with only luminol in the buffer. The periphery of each slice was indicated by broken line. (e); Time course analyses as luminol-mediated CL of the activity of local OXB on HWC-elicitor treated surface of potato tuber slices following stimulation. Each value is the average of integral number of photons generated in a area of 1 cm2 for 5 min at the designated times after stimulation with HWC-elicitor in three experiments. Bars mean the standard deviation from the mean. Solid column: treated with HWC-elicitor, shaded column: treated with buffer only.
two cell layers from the elicitor-treated surface even at 1–2 days after treatment with HWC-elicitor at 1 mg/ml (data not shown). This meant that the HWC-elicitor may not systemically permeate even to 1 mm depth (10 – 15 cells layers) in tissue slices.
3.2. Appearance of sub-systemic OXB on non-treated part of HWC-elicitor treated potato tuber slices Potato tubers were horizontally sliced to stem axis with 3.5 cm thick from top to bottom sur-
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faces. Each was vertically sliced into two halves and then aged for 18 h to develop hypersensitive reactivity of surface cells to HWC-elicitor [12]. A point treatment with 100 ml HWC-elicitor at 1 mg/ml was made by application on 25 mm2 of parenchymatous tissue of the bottom surface of one half. The luminol solution was applied on the whole top surface. CL on the luminol-applied surface was continuously monitored and imaged as photographs (Fig. 2A). The CL initially appeared as scattered small spots at random from 20 to 30 min after treatment with HWC-elicitor (Fig. 2A, b). The CL spots gradually increased in number and each developed with increase in size and strength until at least 60 min after induction treatment (Fig. 2A, c and d). Control slices (buffer treatment) revealed systemic CL spots to a lesser degree at around 60 min after treatment (Fig. 2A, c and d). The systemic CL on the elicitor non-treated surface of the elicitor-treated slice was not observed when HWC-elicitor was applied together with the inhibitor of local OXB, DPI (100 mM) (Fig. 3A and B), catabolic enzymes of H2O2, catalase (100 mg/ml) or radical scavenger, tiron (1 mM) (data not shown). The systemic CL in tissue slices treated with HWC-elicitor was not observed when it was assayed in the presence of the above inhibitors. Intensity of the systemic appearance of CL also varied to some extent due to the nature of each tuber slice or storage age. Quantitative analyses showed that the systemic CL significantly appeared around 20 min after induction treatment in the case of 3.5 cm thick slices and reached the maximum level at around 60 min (Fig. 2B). The tissue surface where the sub-systemic OXB appeared did not show visible changes like tissue browning caused by direct treatment with HWCelicitor.
3.3. Appearance of sub-systemic OXB in a compound leaf The upper surface of leaflets at the middle position of an excised compound leaf was applied with a mixture of HWC-elicitor or buffer (control) by carborundum brushing to produce elicitor
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Fig. 2. Time course observations of a sub-systemic oxidative burst as images of luminol-mediated CL on the HWCelicitor non-treated surface of potato tuber tissues (3.5 cm thick) which were made by horizontal slicing to stem axis (A). The slice was vertically cut into two halves for stimulation with HWC-elicitor (HWC) or buffer (CONT). HWCelicitor was applied by attaching a glass wool disc (0.25 mm2) absorbing HWC-elicitor or buffer solution on the parenchymatous tissues of the bottom. Each upper surface of the slice was entirely applied with luminol solution (2.5 mM). Photons from CL were incorporated for each 10 min at 10 (a), 20 (b), 40 (c) and 60 (d) min after stimulation and imaged as described in Fig. 1. Upper and lower sets shows two typical results. (B) Time course analyses of (A). Each value is the average of integral number of photons generated in area of 1 cm2 for every 5 min at designated times after elicitor treatment in three experiments. Bars mean the standard deviation from the mean. Solid column: treated with HWC-elicitor, shaded column: treated with buffer only.
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on almost the whole surface of HWC-elicitor treated leaflets (Fig. 4A, a). The CL was also observed on the non-treated leaflets at the basal and top position to a significant extent. Buffertreated leaflets locally and systemically showed CL spots only at a lesser level (Fig. 4A, b). The compound leaf which was not pre-treated with
Fig. 3. Effect of DPI on the HWC-induced sub-systemic OXB in potato tuber. (A) Images of luminol-mediated CL of effect of DPI on induction of the HWC-induced systemic OXB. Bottom surface of each disc was treated with HWC (1.0 mg/ml) in 10 mM Tris – HCl buffer (pH 7.4) (a and d); buffer only (b); HWC solution (1.0 mg/ml) containing DPI (100 mM) (c). On the upper surface of each disc, luminol (2.5 mM) containing DPI (d), not containing DPI was applied (a, b and c). CL on the upper surface was imaged by incorporation for 5 min after 60 min. Thickness of the tissue used was 3.5 cm. (B) Quantitative assay as luminol-mediated CL of (A). Each value is the average of integral number of photons generated in a area of 1 cm2 for 5 min at the designated times after stimulation with HWC-elicitor in three experiments. Bars mean the standard deviation from the mean.
effect, while the other leaflets were applied with buffer plus carborundum brushing. One hour after elicitor treatment, the CL on the surface of each leaflet was monitored from 10 min in the dark after application of luminol solution on the surface of each leaflet since a CL derived from quenching of chlorophyll appeared. CL appeared
Fig. 4. Imaging analyses as a function of luminol-mediated CL of local and sub-systemic oxidative burst in leaflets of the compound leaves of potato plant. (A) Upper surface of two leaflets located at middle position of an excised compound leaf were treated with HWC-elicitor by brushing with carborundum and other leaflets were only brushed with buffer containing carborundum. Upper surface of all leaflets was applied with luminol 1 h after elicitor treatment, kept in the dark for 10 min, and then subjected to imaging by integral counting of photons for 10 min in the dark. White spots in leaflets mean the area showing CL. (a) The middle leaflets treated with elicitor; (b) the middle leaflets treated with buffer; and (c) all leaflet treated with nothing. Outline of each leaflet was drawn by broken line. (B) Quantitative assay of the sub-systemic OXB in HWC-elicitor non-treated leaflets at top and basal parts of a compound leaf, two leaflets at the middle position of which were treated with HWC-elicitor. Experimental systems and methods were the same as for (A). Each value means the average percent of the systemically appeared CL level to the level of locally appeared CL in elicitor-treated leaflets in three experiments. Bars mean the standard deviation from the mean.
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carborundum scarcely showed CL on each leaflets even if the two leaflets at middle position was applied with elicitor solution (Fig. 4A, c). The intensity of the CL level varied from sample to sample. Thus, percent value of the CL in HWC-elicitor non-treated leaflets to that in the elicitortreated leaflets was calculated in each sample for a comparison of the systematically appeared CL level. At 1 h after HWC-elicitor treatment, the levels of CL on the non-treated leaflets at the basal and top parts reached to about 29 and 34%, respectively (Fig. 4B). These observations indicated that HWC-elicitor treatment caused the OXB not only on directly treated leaflets but also on nontreated leaflets to both directions within 1 h.
3.4. Analyses of two-dimensional mo6ement of the sub-systemic OXB in a potato tuber slice A spot treatment with HWC-elicitor solution was done by attaching a small glass wool disk absorbing HWC-elicitor solution in the center of horizontally sliced tuber tissue (long axis: 4 – 5 cm, and short axis: 3–4 cm). The remaining slice surface was covered by spreading with luminol solution only. A time course charge of the development of CL showed an initial appearance of CL as scattered spots at random on the luminol-treated area from about 10 min after HWC-elicitor treatment (Fig. 5, b). The CL spots developed much stronger and larger with incubation time, and spread on almost the whole surface until about 40 min after the spot treatment (Fig. 5c and d). Buffer-treated tissues revealed a few CL spots at around 40 min. This indicated that elicitor treatment at the center of parenchymatous tissues of horizontally sliced tissues developed the sub-systemic OXB to vertical direction. Distinguished difference in the CL appearance was not observed among parenchymatous, vascular bundle and cortex tissues. A spot treatment with HWC-elicitor was done on the edge at the basal part of vertically sliced tuber tissues (10 mm thick, longitudinal stem axis: 8 – 9 cm long) and the luminol solution was uniformly applied on the whole slice-surface (Fig. 6A, a). Time-sequential observations of the CL development showed an initiation of the CL near the HWC-elicitor-treated part from about 10 min after
Fig. 5. Time course imaging of the sub-systemic OXB as the luminol-mediated CL in horizontally sliced tissues (1 cm thick) in the center of which a spot treatment with HWC-elicitor was done. The elicitor non-treated area was applied with luminol solution. Photons from the CL were incorporated for each 5 min at 10 (a), 20 (b), 30 (c) and 40 (d) min after elicitor treatment. White area on the slice surface means the CL generating area. Periphery of each slice was drawn by the broken line.
treatment. The CL area gradually and radialy developed to the direction of the top area of the tuber slice with a gradient of CL intensity from the basal area. At 60 min after treatment, the top edge of CL area reached nearly half of the tuber slices at the elicitor-treated site (Fig. 6A). At the front of CL area, scattered CL spots appeared during the development. No distinguished appearance of the CL was observed among parenchymatous, vascular bundle and cortex tissues on the sliced surface. At late stages, it was observed that buffer treatment caused the CL development near treated site to a lesser extent (Fig. 6A). From repeated experiments, time course changes in the average values of CL were analyzed in each spot (about 0.25 cm2) at definite distance from the HWC-elicitor treated site. The CL level at the nearest spot increased most strongly with a sigmoidal curve with time (Fig.
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Fig. 6. Time course imaging of the sub-systemic OXB on vertically sliced tissues of potato tuber at the edge of the basal part of which a point treatment with HWC-elicitor was done (A). Luminol solution was spread on the whole surface of the slice (long axis: 8 cm, 10 mm thick). Each image was designated by incorporation of photons generated for 5 min from 10 (b), 20 (c), 30 (d), 40 (e) or 60 (f) min after elicitor treatment. Light blue, yellow, yellow red and white color on the slices mean the area of CL generation and its intensity in that order. Periphery of each slice was drawn by broken line. (B) Time course change in the activity of luminol mediated CL by sub-systemic OXB on vertically sliced tuber tissues at the edge of the basal part of which a point treatment with HWC-elicitor was done. Experimental systems and methods were the same as for (A). The counting spots were set at every 2 cm distant from spot 3 just neighboring to the elicitor treated spot. Each value represents the average value of three experiments showing integral number of photons at each spot designated in inlet figures for each 5 min from every 5 min after elicitor treatment.
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6B). The CL at each spot distant by 2 cm from the basal spot also showed similar increasing curve, but at a half level of that of the basal spot area. The CL at each spot distant by 4 cm from the basal spot was much more lower (about 1/10 of CL level at the basal spot area). (Fig. 6B). Within 60 min after HWC-elicitor treatment, the detectable CL reached to at least 4 or 5 cm from the elicitor-treated site. This indicated that local OXB developed a sub-systemic OXB in a tuber stem tissue to a direction from bottom to top with time.
4. Discussion A hypothesis that the OXB in plant cells caused by direct treatment with HWC-elicitor (local OXB) may trigger a sub-systemic OXB at distance from initially activated cells was investigated using potato tuber tissues (stem tissue). The local and sub-systemic OXB were determined by timesequential and two-dimensional imaging analyses of the luminol-mediated CL due to active oxygen species generated on the surface of tissue-slices. Experimental results evidently demonstrated a development of the CL to tissue area distant from the site with the local OXB caused by direct contact with HWC-elicitor (Fig. 1). Elicitor-stimulated CL on plant tissues [13] or suspension-cultured cells [17] have been shown to be predominantly due to H2O2 since concomitant presence of catalase with luminol diminished the CL appearance [13,18]. Additionally, protoplasts without cell wall rapidly activated a SOD-sensitive extra-cellular cytochrome c reducing activity upon reaction with elicitor, suggesting O2− production outside of protoplasts [19]. Elicitor-stimulated O2− generation in plant cells has been proposed to be dependent on an O2− generating NADPH oxidase in plasma membrane [19,20]. The catalase-inhibitory CL was inhibited by a known inhibitor of O2− generating NADPH oxidase, DPI [27]. The systemically appeared CL may be due to an activation of O2− generating NADPH oxidase similar to that in local OXB since it was also inhibited by DPI (Fig. 3).
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A possibility that H2O2 generated during local OXB moves to a distant place through intercellular space of the tuber tissue by diffusion or capillary movement and causes the sub-systemic CL could be ruled out because of the following reasons. The sub-systemic appearance of CL was also inhibited by DPI. If H2O2 systemically moves to the opposite surface at 3.5 cm distance, DPI could not inhibit the appearance of sub-systemic CL. Furthermore, if HWC-elicitor itself systemically reached to a distant place, browned tissue cells could not be restricted in one or two cell layers from HWC elicitor-treated surface. Our preliminary experiments also showed that other inhibitors of the local OXB such as Ca2 + chelator, EGTA, protein kinase inhibitor, staurosporine etc. inhibited the induction of sub-systemic OXB [10]. The system for the subsystemic OXB could be similar to that for the local OXB. Why and by what the sub-systemic OXB is activated remains an interesting subject in relation to systemic signaling in plant tissues. The local and sub-systemic OXB were interestingly characterized by initial appearance of scattered spots in tissues (Fig. 1A and 2A). The CL area developed further by enlarging of each CL spot and increasing the spot number to the whole tissue surface. It seemed that some characteristic cells in the tissues may sensitively recognize the elicitor or initiate the active oxygen generating reaction following treatment with HWC-elicitor. In the case of sub-systemic OXB, however, some cells may be assumed to accept a systemic signal from the original cells with OXB other than elicitor molecule itself. The initial CL spots in sub-systemic OXB became like cores for further development of the CL to neighboring cells through cell to cell. This assumption raises a possibility that specific cells responsible for the initial OXB may be differentiated in the potato tuber tissues and that there may be some systemic signals which could activate the OXB from cell to cell. It is also interesting to consider the previous observations that potato tuber tissues provided both protoplasts with and without hypersensitive reactivity to HWC-elicitor [16]. What signal system may be involved in the induction of sub-systemic OXB is obscure in the
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present experiment. The data showed that inhibition of the induction of the local OXB by DPI (Fig. 3A and 3B) or scavengers of generated active oxygen, catalase or tiron resulted in little appearance of the sub-systemic OXB. So, we assumed that the sub-systemic OXB could be triggered by unknown mobile signal system stimulated by active oxygen species generated during local OXB. The present experiments showed that inhibition of the local CL in the presence of catalase resulted in less appearance of the sub-systemic CL. These preliminary results suggested that the sub-systemic CL may be triggered mainly by H2O2 generated during the local OXB, which is dismutated from O2− generated by the NADPH oxidase. Our preliminary experiments also showed that exogeneously applied H2O2 solution caused an induction of sub-systemic CL in potato tuber tissues [10]. Therefore, H2O2 produced in the local OXB could be a potential trigger of the sub-systemic OXB. The mechanisms of development of the OXB from cell to cell remain to be clarified in relation to the systemic signaling for the response against elicitor in plant tissues. The present experiment using excised compound leaves showed a possibility of the systemic OXB between leaflets in a compound leaf (Fig. 4A and 4B). Application of a mixture of HWC-elicitor and luminol solutions on the surface of leaflets of excised compound leaves caused a CL, indicating the induction of local OXB in leaf tissues (Fig. 4A, a). A treatment of two leaflets at middle position of a compound leaf with HWC-elicitor also showed a sub-systemic CL on the other leaflets treated only with luminol (Fig. 4A, b). This suggested an appearance of sub-systemic OXB in excised compound leaf. The present observations of local and sub-systemic OXB among the leaflets of excised compound leaves may be connected to induction of a possible systemic OXB in whole plants as shown previously [23] through petiole from leaflets and the main stem from compound leaves. The time-sequential observation of sub-systemic OXB in vertically sliced tuber tissues (Fig. 6B) suggested a possibility of subsequent development of OXB from cell to cell in stem tissues. It is assumed that some signals for sub-systemic OXB
may move through the main stem of potato plant. Since the present experiments allowed a continuous observation of CL for only 1 h after elicitorstimulation, movement of the OXB to the distant part was confirmed by no more than 5 cm during observation. It is necessary to devise some methods for determination of OXB for longer periods and distance in the whole plant. Application of HWC-elicitor and luminol solutions on leaflets was done by brushing with carborundum since there is little response of epidermal cells to these agents without a wound. Without carborundum-wounding, little CL was observed even in leaf tissues applied with HWCelicitor. The cuticle layer of leaflets seems to reject HWC-elicitor or luminol to the reaction site in tissue cells. The observation of CL in the leaflet was done for a limited time from 10 min after starting the CL observation in the dark. It was because plant tissues containing chlorophyll reflect a CL for several minutes immediately after moving from light to dark conditions. The CL due to the local OXB in leaflets tended to decline with increase in incubation time for several minutes in the dark. The decline of the elicitor-stimulated CL can not be recovered in the dark since NADPH generated during photosynthesis could be possibly associated with the OXB. The elicitorstimulated OXB in potato plant leaflets had been detected as SOD-sensitive reduction of cytochrome c in the light [7]. As potato tuber tissues provide NADPH through activated pentose mono-phoshate shunt in the course of resistance response [24], the CL seems to continue for a long time in the dark. In the present experimental systems, we did not determine the induction of SAR in relation to induction of sub-systemic OXB since potato tuber (storage stem) slices and detached compound leaves were not appropriate for determination of SAR. Local treatment of leaves of potato plants with HWC-elicitor had been shown to induce a systemic OXB [7] as well as SAR against P. infestans [8]. Furthermore, potato plants grown from potato tuber treated with HWC-elicitor had been shown to acquire a SAR [25,26]. In association with these reports, it is assumed that local OXB could trigger the systemic OXB and SAR in
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potato plants, in which an inducer of SAR did not induce systemic synthesis of salicylic acid [5]. The present paper only demonstrated an induction of novel sub-systemic OXB by inducer of both local OXB and SAR in restricted tissues of potato plants.
Acknowledgements The authors appreciate Drs H. Yoshioka and T. Tsuge for helpful discussion and Y. Umemura for supplying potato tuber seeds. The first author acknowledges the scholarship grants from Yoneyama Rotary Foundation (1994 – 1996) and the Ministry of Education, Science and Culture of Japan (1996–1998). This work was partly supported by grant-in-aid for Exploratory Research (No. 09876092) and for Scientific Research (B) (No. 09460027) from the Ministry of Education, Science and Culture.
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