- Email: [email protected]
Reactive oxygen and ethylene are involved in the regulation of regurgitant-induced responses in bean plants
J. Plant Physiol. 161. 191 – 196 (2004) http://www.elsevier-deutschland.de/jplhp
Reactive oxygen and ethylene are involved in the regulation of regurgitant-induced responses in bean plants Ineta Steinite, Agnese Gailite, Gederts Ievinsh* Department of Plant Physiology, Faculty of Biology, University of Latvia, 4 Kronvalda Blv., Riga LV-1010, Latvia Received March 10, 2003 · Accepted May 19, 2003
Summary Application of regurgitant from Leptinotarsa decemlineata Say on wound surfaces of one wounded leaf of intact bean (Phaseolus vulgaris L.) plants resulted in activation of ethylene biosynthesis followed by an increase of both peroxidase and polyphenol oxidase activity. The aim of the present investigation was to study the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants. As the regurgitant contained relative high activities of both peroxidase and polyphenol oxidase, there is a possibility that increased enzyme activities in bean leaves following regurgitant treatment is an artifact of insectderived enzymes. Localisation experiments and electrophoretic analysis revealed that only part of the increased enzyme activities could be attributed to regurgitant-derived enzymes. Both increase of ethylene production and oxidative enzyme activities depended on protein synthesis. To demonstrate if the increase of oxidative metabolism was ethylene-dependent, seedlings were pretreated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, and 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. Increase of both peroxidase and polyphenol oxidase activity in wounded and subsequently regurgitant-treated leaf was abolished by both aminooxyacetic acid and 1-MCP. Inhibitor studies indicated that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for regurgitant-induced increase of ethylene production and oxidative enzyme activities. Key words: ethylene – herbivores – Leptinotarsa decemlineata – peroxidase – Phaseolus vulgaris – polyphenol oxidase – reactive oxygen species – regurgitant Abbreviations: 1-MCP = 1-methylcyclopropene
Introduction Recognition of herbivore attack on plants is an emerging topic in biological science (Kessler and Baldwin 2002). It is * E-mail corresponding author: [email protected]
already generally accepted that herbivory results in plant responses different from those induced by tissue wounding. Both amplification and inhibition of defense responses can be suggested, depending on the type of attacking herbivore. It was suggested recently that ethylene plays a central role in tailoring herbivore-induced responses to maximise their 0176-1617/04/161/02-191 $ 30.00/0
192
Ineta Steinite, Agnese Gailite, Gederts Ievinsh
defensive function (Kahl et al. 2000). Similarly, ethylene is thought to amplify jasmonate-dependent wound signal during wound-induced responses (Rojo et al. 1999). However, upstream events of herbivore recognition are not well characterised. During pathogenesis and wounding, plasma-membrane NADPH oxidase is involved in generation of reactive oxygen species acting as endogenous signals for induction of defenseresponses (Grant and Loake 2000, Orozco-Cardenas and Ryan 1999, Orozco-Cardenas et al. 2001). It might be suggested that reactive oxygen species act as intermediates during herbivore-derived signal recognition as well. However, it has been argued that reactive oxygen species, which are necessary predecessors of hypersensitive response during pathogen attack, may not be involved in regulation of plantherbivore interactions (Kessler and Baldwin 2002). Our previous experiments have shown that the application of regurgitant from larvae of Leptinotarsa decemlineata caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants (Kruzmane et al. 2002). In addition, hydrogen peroxide appeared as a potential signal of ethylene formation in wounded leaves. The present experiments were designed to analyze the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants.
Materials and Methods Seeds of Phaseolus vulgaris L. cv. Saxa bush bean were surface sterilized with KMnO4 and sown individually into 10-cm plastic pots containing commercial peat-moss (pH 6.0) with addition of mineral nutrients. Plants at a two-leaf stage with fully grown primary leaves were used for experiments. Plants were grown in a growth chamber with a photoperiod of 16/ 8 h in a photosynthetic photon flux density of 120 µmol m – 2 s –1, 25 ± 2 ˚C, 50 to 70 % relative humidity. For pre-treatment with inhibitors, bean plants were de-rooted and individually incubated in glass flasks containing appropriate inhibitor solution for 3 h. The final concentrations in the medium for inhibitors were as follows: 50 µmol L –1 cycloheximide (translation inhibitor), 2 mmol L –1 aminooxyacetic acid (inhibitor of ethylene biosynthesis), 10 mmol L –1 imidazole (inhibitor of cytochrome b component of plasma-membrane NADPH oxidase), 10 µmol L –1 diethyldithiocarbamate (inhibitor of Cu/Zn-superoxide dismutase; Heikkila et al. 1976). Control plants were incubated in water. For 1-methylcyclopropene (1-MCP, irreversible inhibitor of ethylene binding) pre-treatment, plants were placed inside of a 5-L polystyrene chamber 12 h before wounding and/or regurgitant treatment were started. To release 1-MCP into a chamber, 10 mg of SmartFreshTM (AgroFresh Inc., USA) was placed in a vial containing 5 mL of water. Eggs of L. decemlineata were obtained from the Federal Biological Research Center for Agriculture and Forestry, Institute for Biological Control, Darmstadt, Germany. Larvae were reared on potato plants. Regurgitant was collected from the oral cavity of 4-th instar larvae with capillary tubes connected to a vacuum and stored at – 80 ˚C until use.
For regurgitant treatment, one leaf of a pair was wounded with a razor blade making cuts between main veins. A total length of wound surface was 150 mm. Regurgitant was applied to wound surfaces using a glass capillary. About 5 µL of regurgitant was used for 10 mm of wound surface. For localisation experiments, discs (5 mm diameter) were removed with a cork borer from wounded leaves as shown in the inset of Figure 1. Appropriate discs were pooled and used as a source for enzyme extraction. For inhibitor experiments, a central part of a leaf (zone A) containing applied regurgitant, was dissected and discarded. Ethylene measurement and enzyme extraction and assays were carried out as described previously (Kruzmane et al. 2002). Briefly, for measurement of the basal rate of ethylene production, detached leaves were rinsed with water, blotted dry, cut in half, rolled and placed individually in 4-mL screw-capped bottles. After 20 min, 1 mL of headspace gas was analyzed for ethylene concentration using a gas chromatograph Chrom 5 (Czech Republic), equipped with a glass column filled with Al2O3 and a flame ionization detector. Helium was used as a carrier gas. Ethylene content was calculated according to standard curve. For extraction of enzymes, leaf tissues were ground in liquid nitrogen and extracted with 25 mmol L –1 HEPES (pH 7.2) for 15 min at 4 ˚C. The homogenate was filtered through nylon cloth and centrifuged at 15,000 gn for 15 min. Peroxidase (EC 1.11.1.7) activity was measured in a reaction mixture (3 mL) containing 0.05 mol L –1 potassium phosphate (pH 7.0), 15 mmol L –1 guaiacol, 0.03 mol L –1 of hydrogen peroxide, and 20 µL of the enzyme extract. The activity was determined by monitoring the increase in absorbance at 470 nm following tetraguaiacol formation (Maehly and Chance 1954). Diphenolase (EC 1.10.3.2) activity of polyphenol oxidase was measured according to Gauillard et al. (1993). The reaction mixture (3 mL) contained 25 mmol L –1 pyrocatechol in 0.1 mol L –1 potassium phosphate (pH 6.5), and 10 µL of the enzyme extract. The increase in absorbance at 410 nm was monitored. Regurgitant from L. decemlineata and bean leaf extracts were subjected to native polyacrylamide gel electrophoresis. Separation was performed on Hoefer minigel system (Amersham Biosciences) at 4 ˚C, using 7.5 % polyacrylamide gel in anionic system. Protein zones with peroxidase activity in the gels were visualised by incubation in 0.04 % (w/v) benzidine containing 25 mmol L –1 H2O2. All experiments were repeated three times, with similar results. Only results from a representative experiment are presented. Data are expressed as a mean from three to four identical samples ± SE. All the calculations (means and standard error) were performed using MS Excel. Students’ t-test was used to determine the levels of significance. All chemicals were from Sigma-Aldrich Chemie, Germany.
Results and Discussion Localisation of increased enzyme activities Herbivore saliva is known to contain oxidative enzymes, e.g., peroxidase and polyphenol oxidase (Felton and Eischenseer 1999). Therefore, we tested a possibility that increased peroxidase and polyphenol oxidase activity in bean leaves treated with regurgitant from L. decemlineata (Kruzmane et al. 2002) is an artifact due to insect-derived enzymes. Indeed, peroxidase and polyphenol oxidase activities found in
Regulation of regurgitant-induced responses
193
Figure 1. Effect of regurgitant from L. decemlineata larvae on peroxidase activity in different leaf zones of intact bean plants. One leaf of intact bean plant was wounded with a razor blade and regurgitant was applied to wound surfaces by a glass capillary. After appropriate intervals of time leaves were excised. Discs were removed with a cork borer from the appropriate zones as shown in the inset and used as a material for enzyme analysis. A, central part of the leaf, which was wounded and contained applied regurgitant; B, C, D, unwounded zones towards both edges of the leaf. Discs from appropriate zones B, C, D from appropriate leaf were pooled. Dashed line represents the wound. Data are means from 3 samples ± SE at every time point.
the regurgitant from L. decemlineata larvae appeared to affect the level of oxidative enzyme activities measured in regurgitant-treated bean leaves. The regurgitant from L. decemlineata contained extremely high activity of peroxidase as well as the activity of polyphenol oxidase (data not shown). Therefore, localisation of enzyme activities in different zones of regurgitant-treated bean leaf was measured. If regurgitantderived activities of peroxidase and polyphenol oxidase were the only source of the increased activities in bean leaves after the regurgitant treatment, this increase could only be found in a vicinity near to the wounded tissues. However, this was not the case. Regurgitant-dependent increase of enzyme activities was found in more distant leaf tissues as well (Figs. 1 and 2). Though, peroxidase activity only transiently increased in zones C and D (Figs. 1 C, D), there was no statistically significant increase of polyphenol oxidase activity due to the regur-
gitant treatment in these zones (Figs. 2 C, D). In addition, there was a strong dependence of increased activities of enzymes on protein synthesis in unwounded leaf tissues of regurgitanttreated plants (Table 1). Electrophoretic analysis clearly revealed that the regurgitant contains peroxidase isozyme present also in wounded and subsequently regurgitant-treated zone of bean leaves (Fig. 3). This particular peroxidase isozyme was not found in the other leaf zones. Only anionic peroxidases were found to be present in the regurgitant. In addition, stimulation of beanspecific peroxidase isozyme was evident in zone A (Fig. 3).
Effect of inhibitors on regurgitant-induced responses Bean plants were pre-treated with different inhibitors to test the necessity of protein synthesis, ethylene synthesis and
194
Ineta Steinite, Agnese Gailite, Gederts Ievinsh
Figure 2. Effect of regurgitant from L. decemlineata larvae on polyphenol oxidase activity in different leaf zones of intact bean plants. One leaf of intact bean plant was wounded with a razor blade and regurgitant was applied to wound surfaces by a glass capillary. After appropriate intervals of time leaves were excised. Discs were removed with a cork borer from the appropriate zones as shown in the inset for Fig. 1 and used as a material for enzyme analysis. Data are means from 3 samples ± SE at every time point.
Table 1. Effect of inhibitors on regurgitant-induced ethylene production, peroxidase and polyphenol oxidase activity. For 1-MCP pretreatment, plants were placed inside of a 5-L polystyrene chamber 12 h before regurgitant treatment was started. To release MCP into a chamber, 10 mg of SmartFreshTM was placed in a vial containing 5 mL of water. For all other pretreatments, detached bean plants were incubated in appropriate solution of inhibitors for 3 h before regurgitant treatment. The final concentrations in the medium for inhibitors were as follows: 50 µmol L –1 cycloheximide, 2 mmol L –1 aminooxyacetic acid, 10 mmol L –1 imidazole, 10 µmol L –1 diethyldithiocarbamate. For enzyme extraction, only unwounded leaf zones of regurgitant-treated plants were used (zones B, C and D; as in Fig. 1). Increase of parameters in leaves of regurgitant-treated bean plants over control level (wounded leaves) was designated as 100 %. Data are means from 3 independent experiments with 3 replicates each. Values followed by the same letter within a column indicate no significant differences for p = 0.05.
Regurgitant Cycloheximide + regurgitant Aminooxyacetic acid + regurgitant 1-MCP + regurgitant Imidazole + regurgitant Diethyldithiocarbamate + regurgitant
Ethylene (%)
Peroxidase activity (%)
Polyphenol oxidase activity (%)
100a 3b 10c 110a 46d 37d
100a 7b 9b 7b 9b 12b
100a 12b 7b 10b 9b 7b
Regulation of regurgitant-induced responses
Figure 3. Electrophoretic pattern of anionic peroxidases in regurgitant of L. decemlineata (RG) and tissue extracts from different zones (A and B, as in the inset for Fig. 1) of wounded and subsequently regurgitant-treated bean leaf.
perception, and active oxygen species for the regurgitantdependent responses. The translation inhibitor cycloheximide completely inhibited the increase of ethylene production due to regurgitant application (Table 1). Similarly, enzyme activities were significantly inhibited by the cycloheximide (by 93 % and 88 %, for peroxidase and polyphenol oxidase, respectively). Aminooxyacetic acid pretreatment reduced the increase in peroxidase activity and polyphenol oxidase activity by 91% and 93 %, respectively, which corresponded to the level of inhibition of ethylene production by aminooxyacetic acid (90 %, Table 1). When bean plants were pre-treated with 1-MCP, resulting in irreversible blocking of ethylene receptors, regurgitant-induced enzyme activities were inhibited by 93 % and 90 %, for peroxidase and polyphenol oxidase, respectively (Table 1). Ethylene production in regurgitant-treated bean plants (Table 1) as well as in control plants was not significantly affected by 1-MCP treatment (data not shown). Imidazole, which binds to the cytochrome b component of plasma-membrane NADPH oxidase, suppressed regurgitantdependent ethylene production by 54 % (Table 1). Imidazole pre-treatment resulted in significant reduction of both enzyme activities by 91 %. Diethyldithiocarbamate, an inhibitor of Cu/Zn-superoxide dismutase (Heikkila et al. 1976), was used to determine if H2O2 formation through dismutation of O2˙ – by superoxide dismutase is necessary for regurgitant-induced responses. In diethyldithiocarbamate-treated bean plants, increase of ethylene production by regurgitant was suppressed by 63 %, but increase in peroxidase and polyphenol oxidase activities – by 89 % and 93 %, respectively (Table 1). It is generally accepted that the NADPH oxidase is responsible for much of the reactive oxygen species produced after pathogen infection in Arabidopsis (Torres et al. 2001). However, it was shown, that the major source of reactive oxygen species during elicitation of defense responses in bean seedlings is dependent on peroxidase (Bolwell et al. 2002). It seems that in wounded bean leaves application of regurgitant from L. decemlineata leads to activation of different pathways than those during pathogen responses. It should be men-
195
tioned that the role of NADPH oxidase as a generator of intracellular signals has evolved during other abiotic stressinduced antioxidant responses apart from tissue wounding (Jiang and Zhang 2002). In these experiments, treatment with NADPH oxidase inhibitor imidazole significantly reduced the increase in the activities of antioxidant enzymes induced by water stress. In contrast to studies where O2˙ – rather than H2O2 was shown to be involved in activation of defense responses (Jabs et al. 1997), in bean leaves inhibitor of superoxide dismutase strongly suppressed activation of oxidative enzymes by the regurgitant (Table 1). This data indicates that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for the regurgitant-induced increase of ethylene production and oxidative enzyme activities. Consequently, the necessity for H2O2 synthesis during regurgitantinduced responses is similar to that described for H2O2 during pathogenesis. Ethylene production was less sensitive to inhibitors of formation of reactive oxygen species than the activities of peroxidase and polyphenol oxidase. However, inhibition of ethylene biosynthesis or blocking the ethylene receptors with 1-MCP resulted in almost complete inhibition of increase in enzyme activities induced by regurgitant. This suggests that other signals, downstream reactive oxygen species, besides ethylene. are involved in the control of oxidative enzyme activities during regurgitant-induced responses in bean plants. The regulative role of ethylene during wound responses as well as during herbivore-dependent responses is controversial. In contrast to solanaceous plants, where ethylene potentiates wound-induced jasmonate and oligosaccharide signals (O’Donnell et al. 1996), ethylene is suggested to suppress jasmonate-induced gene expression in damaged leaves of Arabidopsis (Rojo et al. 1999). Based on 1-MCP experiments, it was concluded that only insect-induced volatile emission, but not jasmonate, increase depended on ethylene perception in Zea mays plants (Schmelz et al. 2003). In Nicotiana attenuata 1-MCP pre-treatment dramatically amplified the transcript accumulation resulting from both wounding and Manduca sexta herbivory indicating that herbivore-induced ethylene is responsible for depression of direct defense responses (Kahl et al. 2000). The defense response, e.a., nicotine synthesis, was clearly of systemic nature, as leaf wounding/treatment caused decrease of nicotine synthesis in roots. In Arabidopsis, ethylene signal transduction pathway had contrasting effects on the herbivory of different insect species (Stotz et al. 2000). Based on these experiments, it seems that the dependence of herbivore-induced responses on ethylene is different in respect to local vs. systemic tissues. This data indicates that ethylene synthesis and perception is necessary for induction of responses locally in damaged leaves of bean plants (Table 1), while in distant non-damaged leaves ethylene suppresses both wound- and regurgitant-induced responses (Ievinsh et al., unpublished data). In conclusion, oxidative burst through NADPH oxidase and superoxide dismutase appears to be involved in regurgitant-
196
Ineta Steinite, Agnese Gailite, Gederts Ievinsh
dependent increase in ethylene production and activities of peroxidase and polyphenol oxidase in bean leaves. Ethylene biosynthesis and perception, in turn, is responsible for the increase of oxidative enzyme activities after regurgitant application. The described responses clearly represent general defense reactions of plants to regurgitant of non-specific herbivorous insects (Kruzmane et al. 2002). Acknowledgements. This work was supported by the Science Council of Latvia (grant # 01.0248). We thank Mr. H. Warner (AgroFresh Inc., USA) for the gift of SmartFreshTM. We also thank Gaida Arente for assistance with electrophoresis.
References Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F (2002) The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot 53: 1367–1376 Felton GW, Eichenseer H (1999) Herbivore saliva and its effects on plant defense against herbivores and pathogens. In: Agrawal AA et al (eds) Induced Plant Defenses Against Pathogens and Herbivores. APS Press, St. Paul, MN, pp 19 – 36 Gauillard F, Richard-Forget F, Nicolas J (1993) New spectrophotometric assay for polyphenol oxidase activity. Anal Biochem 215: 59 – 65 Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124: 21– 29 Heikkila RE, Cabbat FS, Cohen G (1976) In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate. J Biol Chem 251: 2182 – 2185 Jabs T, Tschöpe M, Colling C, Hahlbrock K, Scheel D (1997) Elicitorstimulated ion fluxes and O2 – from the oxidative burst are essential components in triggering defense gene activation and phytoalexin sythesis in parsley. Proc Natl Acad Sci USA 94: 4800 – 4805 Jiang M, Zhang J (2002) Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215: 1022–1030
Kahl J, Siemens DH, Aerts RJ, Gabler R, Kuhnemann F, Preston CA, Baldwin IT (2000) Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore. Planta 210: 336 – 342 Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53: 299 – 328 Kruzmane D, Jankevica L, Ievinsh G (2002) Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris. Physiol Plant 115: 577– 584 Maehly AC, Chance B (1954) The assay of catalases and peroxidases. In: Glick D (ed) Methods of Biochemical Analysis I, pp 357– 424. Interscience Publ, Inc, New York O’Donnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Bowles DJ (1996) Ethylene as a signal mediating the wound response of tomato plants. Science 274: 1914–1917 Orozco-Cardenas ML, Narvaez-Vasquez J, Ryan CA (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13: 179–191 Orozco-Cardenas M, Ryan CA (1999) Hydrogen peroxide is generated systemically in plant leaves by wounding and system in via the octadecanoid pathway. Proc Natl Acad Sci USA 96: 6553 – 6557 Rojo E, Leon J, Sanchez-Serrano JJ (1999) Cross-talk between wound signaling pathways determines local versus systemic gene expression in Arabidopsis thaliana. Plant J 20: 135–142 Schmelz EA, Alborn HT, Banchio E, Tumlinson JH (2003) Quantitative relationships between induced jasmonic acid levels and volatile emission in Zea mays during Spodoptera exigua herbivory. Planta 216: 665 – 673 Stotz HU, Pittendrigh BR, Kroymann J, Weniger K, Fritsche J, Bauke A, Mitchell-Olds T (2000) Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not diamondback moth. Plant Physiol 124: 1007–1017 Torres MA, Dangl JL, Jones JDG (2001) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 98: 517– 522
Reactive oxygen and ethylene are involved in the regulation of regurgitant-induced responses in bean plants Ineta Steinite, Agnese Gailite, Gederts Ievinsh* Department of Plant Physiology, Faculty of Biology, University of Latvia, 4 Kronvalda Blv., Riga LV-1010, Latvia Received March 10, 2003 · Accepted May 19, 2003
Summary Application of regurgitant from Leptinotarsa decemlineata Say on wound surfaces of one wounded leaf of intact bean (Phaseolus vulgaris L.) plants resulted in activation of ethylene biosynthesis followed by an increase of both peroxidase and polyphenol oxidase activity. The aim of the present investigation was to study the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants. As the regurgitant contained relative high activities of both peroxidase and polyphenol oxidase, there is a possibility that increased enzyme activities in bean leaves following regurgitant treatment is an artifact of insectderived enzymes. Localisation experiments and electrophoretic analysis revealed that only part of the increased enzyme activities could be attributed to regurgitant-derived enzymes. Both increase of ethylene production and oxidative enzyme activities depended on protein synthesis. To demonstrate if the increase of oxidative metabolism was ethylene-dependent, seedlings were pretreated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, and 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. Increase of both peroxidase and polyphenol oxidase activity in wounded and subsequently regurgitant-treated leaf was abolished by both aminooxyacetic acid and 1-MCP. Inhibitor studies indicated that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for regurgitant-induced increase of ethylene production and oxidative enzyme activities. Key words: ethylene – herbivores – Leptinotarsa decemlineata – peroxidase – Phaseolus vulgaris – polyphenol oxidase – reactive oxygen species – regurgitant Abbreviations: 1-MCP = 1-methylcyclopropene
Introduction Recognition of herbivore attack on plants is an emerging topic in biological science (Kessler and Baldwin 2002). It is * E-mail corresponding author: [email protected]
already generally accepted that herbivory results in plant responses different from those induced by tissue wounding. Both amplification and inhibition of defense responses can be suggested, depending on the type of attacking herbivore. It was suggested recently that ethylene plays a central role in tailoring herbivore-induced responses to maximise their 0176-1617/04/161/02-191 $ 30.00/0
192
Ineta Steinite, Agnese Gailite, Gederts Ievinsh
defensive function (Kahl et al. 2000). Similarly, ethylene is thought to amplify jasmonate-dependent wound signal during wound-induced responses (Rojo et al. 1999). However, upstream events of herbivore recognition are not well characterised. During pathogenesis and wounding, plasma-membrane NADPH oxidase is involved in generation of reactive oxygen species acting as endogenous signals for induction of defenseresponses (Grant and Loake 2000, Orozco-Cardenas and Ryan 1999, Orozco-Cardenas et al. 2001). It might be suggested that reactive oxygen species act as intermediates during herbivore-derived signal recognition as well. However, it has been argued that reactive oxygen species, which are necessary predecessors of hypersensitive response during pathogen attack, may not be involved in regulation of plantherbivore interactions (Kessler and Baldwin 2002). Our previous experiments have shown that the application of regurgitant from larvae of Leptinotarsa decemlineata caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants (Kruzmane et al. 2002). In addition, hydrogen peroxide appeared as a potential signal of ethylene formation in wounded leaves. The present experiments were designed to analyze the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants.
Materials and Methods Seeds of Phaseolus vulgaris L. cv. Saxa bush bean were surface sterilized with KMnO4 and sown individually into 10-cm plastic pots containing commercial peat-moss (pH 6.0) with addition of mineral nutrients. Plants at a two-leaf stage with fully grown primary leaves were used for experiments. Plants were grown in a growth chamber with a photoperiod of 16/ 8 h in a photosynthetic photon flux density of 120 µmol m – 2 s –1, 25 ± 2 ˚C, 50 to 70 % relative humidity. For pre-treatment with inhibitors, bean plants were de-rooted and individually incubated in glass flasks containing appropriate inhibitor solution for 3 h. The final concentrations in the medium for inhibitors were as follows: 50 µmol L –1 cycloheximide (translation inhibitor), 2 mmol L –1 aminooxyacetic acid (inhibitor of ethylene biosynthesis), 10 mmol L –1 imidazole (inhibitor of cytochrome b component of plasma-membrane NADPH oxidase), 10 µmol L –1 diethyldithiocarbamate (inhibitor of Cu/Zn-superoxide dismutase; Heikkila et al. 1976). Control plants were incubated in water. For 1-methylcyclopropene (1-MCP, irreversible inhibitor of ethylene binding) pre-treatment, plants were placed inside of a 5-L polystyrene chamber 12 h before wounding and/or regurgitant treatment were started. To release 1-MCP into a chamber, 10 mg of SmartFreshTM (AgroFresh Inc., USA) was placed in a vial containing 5 mL of water. Eggs of L. decemlineata were obtained from the Federal Biological Research Center for Agriculture and Forestry, Institute for Biological Control, Darmstadt, Germany. Larvae were reared on potato plants. Regurgitant was collected from the oral cavity of 4-th instar larvae with capillary tubes connected to a vacuum and stored at – 80 ˚C until use.
For regurgitant treatment, one leaf of a pair was wounded with a razor blade making cuts between main veins. A total length of wound surface was 150 mm. Regurgitant was applied to wound surfaces using a glass capillary. About 5 µL of regurgitant was used for 10 mm of wound surface. For localisation experiments, discs (5 mm diameter) were removed with a cork borer from wounded leaves as shown in the inset of Figure 1. Appropriate discs were pooled and used as a source for enzyme extraction. For inhibitor experiments, a central part of a leaf (zone A) containing applied regurgitant, was dissected and discarded. Ethylene measurement and enzyme extraction and assays were carried out as described previously (Kruzmane et al. 2002). Briefly, for measurement of the basal rate of ethylene production, detached leaves were rinsed with water, blotted dry, cut in half, rolled and placed individually in 4-mL screw-capped bottles. After 20 min, 1 mL of headspace gas was analyzed for ethylene concentration using a gas chromatograph Chrom 5 (Czech Republic), equipped with a glass column filled with Al2O3 and a flame ionization detector. Helium was used as a carrier gas. Ethylene content was calculated according to standard curve. For extraction of enzymes, leaf tissues were ground in liquid nitrogen and extracted with 25 mmol L –1 HEPES (pH 7.2) for 15 min at 4 ˚C. The homogenate was filtered through nylon cloth and centrifuged at 15,000 gn for 15 min. Peroxidase (EC 1.11.1.7) activity was measured in a reaction mixture (3 mL) containing 0.05 mol L –1 potassium phosphate (pH 7.0), 15 mmol L –1 guaiacol, 0.03 mol L –1 of hydrogen peroxide, and 20 µL of the enzyme extract. The activity was determined by monitoring the increase in absorbance at 470 nm following tetraguaiacol formation (Maehly and Chance 1954). Diphenolase (EC 1.10.3.2) activity of polyphenol oxidase was measured according to Gauillard et al. (1993). The reaction mixture (3 mL) contained 25 mmol L –1 pyrocatechol in 0.1 mol L –1 potassium phosphate (pH 6.5), and 10 µL of the enzyme extract. The increase in absorbance at 410 nm was monitored. Regurgitant from L. decemlineata and bean leaf extracts were subjected to native polyacrylamide gel electrophoresis. Separation was performed on Hoefer minigel system (Amersham Biosciences) at 4 ˚C, using 7.5 % polyacrylamide gel in anionic system. Protein zones with peroxidase activity in the gels were visualised by incubation in 0.04 % (w/v) benzidine containing 25 mmol L –1 H2O2. All experiments were repeated three times, with similar results. Only results from a representative experiment are presented. Data are expressed as a mean from three to four identical samples ± SE. All the calculations (means and standard error) were performed using MS Excel. Students’ t-test was used to determine the levels of significance. All chemicals were from Sigma-Aldrich Chemie, Germany.
Results and Discussion Localisation of increased enzyme activities Herbivore saliva is known to contain oxidative enzymes, e.g., peroxidase and polyphenol oxidase (Felton and Eischenseer 1999). Therefore, we tested a possibility that increased peroxidase and polyphenol oxidase activity in bean leaves treated with regurgitant from L. decemlineata (Kruzmane et al. 2002) is an artifact due to insect-derived enzymes. Indeed, peroxidase and polyphenol oxidase activities found in
Regulation of regurgitant-induced responses
193
Figure 1. Effect of regurgitant from L. decemlineata larvae on peroxidase activity in different leaf zones of intact bean plants. One leaf of intact bean plant was wounded with a razor blade and regurgitant was applied to wound surfaces by a glass capillary. After appropriate intervals of time leaves were excised. Discs were removed with a cork borer from the appropriate zones as shown in the inset and used as a material for enzyme analysis. A, central part of the leaf, which was wounded and contained applied regurgitant; B, C, D, unwounded zones towards both edges of the leaf. Discs from appropriate zones B, C, D from appropriate leaf were pooled. Dashed line represents the wound. Data are means from 3 samples ± SE at every time point.
the regurgitant from L. decemlineata larvae appeared to affect the level of oxidative enzyme activities measured in regurgitant-treated bean leaves. The regurgitant from L. decemlineata contained extremely high activity of peroxidase as well as the activity of polyphenol oxidase (data not shown). Therefore, localisation of enzyme activities in different zones of regurgitant-treated bean leaf was measured. If regurgitantderived activities of peroxidase and polyphenol oxidase were the only source of the increased activities in bean leaves after the regurgitant treatment, this increase could only be found in a vicinity near to the wounded tissues. However, this was not the case. Regurgitant-dependent increase of enzyme activities was found in more distant leaf tissues as well (Figs. 1 and 2). Though, peroxidase activity only transiently increased in zones C and D (Figs. 1 C, D), there was no statistically significant increase of polyphenol oxidase activity due to the regur-
gitant treatment in these zones (Figs. 2 C, D). In addition, there was a strong dependence of increased activities of enzymes on protein synthesis in unwounded leaf tissues of regurgitanttreated plants (Table 1). Electrophoretic analysis clearly revealed that the regurgitant contains peroxidase isozyme present also in wounded and subsequently regurgitant-treated zone of bean leaves (Fig. 3). This particular peroxidase isozyme was not found in the other leaf zones. Only anionic peroxidases were found to be present in the regurgitant. In addition, stimulation of beanspecific peroxidase isozyme was evident in zone A (Fig. 3).
Effect of inhibitors on regurgitant-induced responses Bean plants were pre-treated with different inhibitors to test the necessity of protein synthesis, ethylene synthesis and
194
Ineta Steinite, Agnese Gailite, Gederts Ievinsh
Figure 2. Effect of regurgitant from L. decemlineata larvae on polyphenol oxidase activity in different leaf zones of intact bean plants. One leaf of intact bean plant was wounded with a razor blade and regurgitant was applied to wound surfaces by a glass capillary. After appropriate intervals of time leaves were excised. Discs were removed with a cork borer from the appropriate zones as shown in the inset for Fig. 1 and used as a material for enzyme analysis. Data are means from 3 samples ± SE at every time point.
Table 1. Effect of inhibitors on regurgitant-induced ethylene production, peroxidase and polyphenol oxidase activity. For 1-MCP pretreatment, plants were placed inside of a 5-L polystyrene chamber 12 h before regurgitant treatment was started. To release MCP into a chamber, 10 mg of SmartFreshTM was placed in a vial containing 5 mL of water. For all other pretreatments, detached bean plants were incubated in appropriate solution of inhibitors for 3 h before regurgitant treatment. The final concentrations in the medium for inhibitors were as follows: 50 µmol L –1 cycloheximide, 2 mmol L –1 aminooxyacetic acid, 10 mmol L –1 imidazole, 10 µmol L –1 diethyldithiocarbamate. For enzyme extraction, only unwounded leaf zones of regurgitant-treated plants were used (zones B, C and D; as in Fig. 1). Increase of parameters in leaves of regurgitant-treated bean plants over control level (wounded leaves) was designated as 100 %. Data are means from 3 independent experiments with 3 replicates each. Values followed by the same letter within a column indicate no significant differences for p = 0.05.
Regurgitant Cycloheximide + regurgitant Aminooxyacetic acid + regurgitant 1-MCP + regurgitant Imidazole + regurgitant Diethyldithiocarbamate + regurgitant
Ethylene (%)
Peroxidase activity (%)
Polyphenol oxidase activity (%)
100a 3b 10c 110a 46d 37d
100a 7b 9b 7b 9b 12b
100a 12b 7b 10b 9b 7b
Regulation of regurgitant-induced responses
Figure 3. Electrophoretic pattern of anionic peroxidases in regurgitant of L. decemlineata (RG) and tissue extracts from different zones (A and B, as in the inset for Fig. 1) of wounded and subsequently regurgitant-treated bean leaf.
perception, and active oxygen species for the regurgitantdependent responses. The translation inhibitor cycloheximide completely inhibited the increase of ethylene production due to regurgitant application (Table 1). Similarly, enzyme activities were significantly inhibited by the cycloheximide (by 93 % and 88 %, for peroxidase and polyphenol oxidase, respectively). Aminooxyacetic acid pretreatment reduced the increase in peroxidase activity and polyphenol oxidase activity by 91% and 93 %, respectively, which corresponded to the level of inhibition of ethylene production by aminooxyacetic acid (90 %, Table 1). When bean plants were pre-treated with 1-MCP, resulting in irreversible blocking of ethylene receptors, regurgitant-induced enzyme activities were inhibited by 93 % and 90 %, for peroxidase and polyphenol oxidase, respectively (Table 1). Ethylene production in regurgitant-treated bean plants (Table 1) as well as in control plants was not significantly affected by 1-MCP treatment (data not shown). Imidazole, which binds to the cytochrome b component of plasma-membrane NADPH oxidase, suppressed regurgitantdependent ethylene production by 54 % (Table 1). Imidazole pre-treatment resulted in significant reduction of both enzyme activities by 91 %. Diethyldithiocarbamate, an inhibitor of Cu/Zn-superoxide dismutase (Heikkila et al. 1976), was used to determine if H2O2 formation through dismutation of O2˙ – by superoxide dismutase is necessary for regurgitant-induced responses. In diethyldithiocarbamate-treated bean plants, increase of ethylene production by regurgitant was suppressed by 63 %, but increase in peroxidase and polyphenol oxidase activities – by 89 % and 93 %, respectively (Table 1). It is generally accepted that the NADPH oxidase is responsible for much of the reactive oxygen species produced after pathogen infection in Arabidopsis (Torres et al. 2001). However, it was shown, that the major source of reactive oxygen species during elicitation of defense responses in bean seedlings is dependent on peroxidase (Bolwell et al. 2002). It seems that in wounded bean leaves application of regurgitant from L. decemlineata leads to activation of different pathways than those during pathogen responses. It should be men-
195
tioned that the role of NADPH oxidase as a generator of intracellular signals has evolved during other abiotic stressinduced antioxidant responses apart from tissue wounding (Jiang and Zhang 2002). In these experiments, treatment with NADPH oxidase inhibitor imidazole significantly reduced the increase in the activities of antioxidant enzymes induced by water stress. In contrast to studies where O2˙ – rather than H2O2 was shown to be involved in activation of defense responses (Jabs et al. 1997), in bean leaves inhibitor of superoxide dismutase strongly suppressed activation of oxidative enzymes by the regurgitant (Table 1). This data indicates that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for the regurgitant-induced increase of ethylene production and oxidative enzyme activities. Consequently, the necessity for H2O2 synthesis during regurgitantinduced responses is similar to that described for H2O2 during pathogenesis. Ethylene production was less sensitive to inhibitors of formation of reactive oxygen species than the activities of peroxidase and polyphenol oxidase. However, inhibition of ethylene biosynthesis or blocking the ethylene receptors with 1-MCP resulted in almost complete inhibition of increase in enzyme activities induced by regurgitant. This suggests that other signals, downstream reactive oxygen species, besides ethylene. are involved in the control of oxidative enzyme activities during regurgitant-induced responses in bean plants. The regulative role of ethylene during wound responses as well as during herbivore-dependent responses is controversial. In contrast to solanaceous plants, where ethylene potentiates wound-induced jasmonate and oligosaccharide signals (O’Donnell et al. 1996), ethylene is suggested to suppress jasmonate-induced gene expression in damaged leaves of Arabidopsis (Rojo et al. 1999). Based on 1-MCP experiments, it was concluded that only insect-induced volatile emission, but not jasmonate, increase depended on ethylene perception in Zea mays plants (Schmelz et al. 2003). In Nicotiana attenuata 1-MCP pre-treatment dramatically amplified the transcript accumulation resulting from both wounding and Manduca sexta herbivory indicating that herbivore-induced ethylene is responsible for depression of direct defense responses (Kahl et al. 2000). The defense response, e.a., nicotine synthesis, was clearly of systemic nature, as leaf wounding/treatment caused decrease of nicotine synthesis in roots. In Arabidopsis, ethylene signal transduction pathway had contrasting effects on the herbivory of different insect species (Stotz et al. 2000). Based on these experiments, it seems that the dependence of herbivore-induced responses on ethylene is different in respect to local vs. systemic tissues. This data indicates that ethylene synthesis and perception is necessary for induction of responses locally in damaged leaves of bean plants (Table 1), while in distant non-damaged leaves ethylene suppresses both wound- and regurgitant-induced responses (Ievinsh et al., unpublished data). In conclusion, oxidative burst through NADPH oxidase and superoxide dismutase appears to be involved in regurgitant-
196
Ineta Steinite, Agnese Gailite, Gederts Ievinsh
dependent increase in ethylene production and activities of peroxidase and polyphenol oxidase in bean leaves. Ethylene biosynthesis and perception, in turn, is responsible for the increase of oxidative enzyme activities after regurgitant application. The described responses clearly represent general defense reactions of plants to regurgitant of non-specific herbivorous insects (Kruzmane et al. 2002). Acknowledgements. This work was supported by the Science Council of Latvia (grant # 01.0248). We thank Mr. H. Warner (AgroFresh Inc., USA) for the gift of SmartFreshTM. We also thank Gaida Arente for assistance with electrophoresis.
References Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F (2002) The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot 53: 1367–1376 Felton GW, Eichenseer H (1999) Herbivore saliva and its effects on plant defense against herbivores and pathogens. In: Agrawal AA et al (eds) Induced Plant Defenses Against Pathogens and Herbivores. APS Press, St. Paul, MN, pp 19 – 36 Gauillard F, Richard-Forget F, Nicolas J (1993) New spectrophotometric assay for polyphenol oxidase activity. Anal Biochem 215: 59 – 65 Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124: 21– 29 Heikkila RE, Cabbat FS, Cohen G (1976) In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate. J Biol Chem 251: 2182 – 2185 Jabs T, Tschöpe M, Colling C, Hahlbrock K, Scheel D (1997) Elicitorstimulated ion fluxes and O2 – from the oxidative burst are essential components in triggering defense gene activation and phytoalexin sythesis in parsley. Proc Natl Acad Sci USA 94: 4800 – 4805 Jiang M, Zhang J (2002) Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215: 1022–1030
Kahl J, Siemens DH, Aerts RJ, Gabler R, Kuhnemann F, Preston CA, Baldwin IT (2000) Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore. Planta 210: 336 – 342 Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53: 299 – 328 Kruzmane D, Jankevica L, Ievinsh G (2002) Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris. Physiol Plant 115: 577– 584 Maehly AC, Chance B (1954) The assay of catalases and peroxidases. In: Glick D (ed) Methods of Biochemical Analysis I, pp 357– 424. Interscience Publ, Inc, New York O’Donnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Bowles DJ (1996) Ethylene as a signal mediating the wound response of tomato plants. Science 274: 1914–1917 Orozco-Cardenas ML, Narvaez-Vasquez J, Ryan CA (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13: 179–191 Orozco-Cardenas M, Ryan CA (1999) Hydrogen peroxide is generated systemically in plant leaves by wounding and system in via the octadecanoid pathway. Proc Natl Acad Sci USA 96: 6553 – 6557 Rojo E, Leon J, Sanchez-Serrano JJ (1999) Cross-talk between wound signaling pathways determines local versus systemic gene expression in Arabidopsis thaliana. Plant J 20: 135–142 Schmelz EA, Alborn HT, Banchio E, Tumlinson JH (2003) Quantitative relationships between induced jasmonic acid levels and volatile emission in Zea mays during Spodoptera exigua herbivory. Planta 216: 665 – 673 Stotz HU, Pittendrigh BR, Kroymann J, Weniger K, Fritsche J, Bauke A, Mitchell-Olds T (2000) Induced plant defense responses against chewing insects. Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not diamondback moth. Plant Physiol 124: 1007–1017 Torres MA, Dangl JL, Jones JDG (2001) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 98: 517– 522