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Catalases negatively regulate methyl jasmonate signaling in guard cells
Journal of Plant Physiology 169 (2012) 1012–1016
Contents lists available at SciVerse ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.de/jplph
Short communication
Catalases negatively regulate methyl jasmonate signaling in guard cells Rayhanur Jannata , Misugi Urajia , Mohammad Anowar Hossaina , Mohammad Muzahidul Islama , Yoshimasa Nakamuraa , Izumi C. Morib,∗ , Yoshiyuki Murataa a b
Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan Institute of Plant Science and Resources, Okayama University, 2-20-1, Kurashiki 710-8530, Japan
a r t i c l e
i n f o
Article history: Received 6 December 2011 Received in revised form 29 March 2012 Accepted 30 March 2012 Keywords: Catalase Guard cells Hydrogen peroxide Methyl jasmonate
a b s t r a c t Methyl jasmonate (MeJA)-induced stomatal closure is accompanied by the accumulation of hydrogen peroxide (H2 O2 ) in guard cells. In this study, we investigated the roles of catalases (CATs) in MeJA-induced stomatal closure using cat mutants cat2, cat3-1 and cat1 cat3, and the CAT inhibitor, 3-aminotriazole (AT). When assessed with 2 ,7 -dichlorodihydrofluorescein, the reduction of catalase activity by means of mutations and the inhibitor accumulated higher basal levels of H2 O2 in guard cells whereas they did not affect stomatal aperture in the absence of MeJA. In contrast, the cat mutations and the treatment with AT potentiated MeJA-induced stomatal closure and MeJA-induced H2 O2 production. These results indicate that CATs negatively regulate H2 O2 accumulation in guard cells and suggest that inducible H2 O2 production rather than constitutive elevation modulates stomatal apertures in Arabidopsis. © 2012 Elsevier GmbH. All rights reserved.
Introduction The stomatal apparatus, which consists of a pair of guard cells in the epidermis of above-ground parts of higher plants, responds to various external and internal stimuli (Schroeder et al., 2001; Hetherington and Woodward, 2003). Stomatal apertures close when exposed to the volatile phytohormone, methyl jasmonate (MeJA), which regulates various physiological processes and reacts to wounding and pathogen attack (Berger et al., 1996; Browse, 2009) by limiting the invasion of pathogens through the pores (Irving et al., 1992; Gehring et al., 1997; Liu et al., 2002; Suhita et al., 2004; Munemasa et al., 2007; Saito et al., 2008; Islam et al., 2010). Hydrogen peroxide (H2 O2 ) is one of the major reactive oxygen species (ROS) and acts as a second messenger in MeJA and abscisic acid (ABA) signal transduction in guard cells. Genetic and pharmacological studies have demonstrated that the ROS production is mediated by NAD(P)H oxidases (Kwak et al., 2003; Suhita et al., 2004). The generated ROS activates plasma membrane Ca2+ permeable channels (ICa channels), which in turn elicit cytosolic free calcium ion concentration ([Ca2+ ]cyt ) elevation/oscillation in guard
Abbreviations: ABA, abscisic acid; APX, ascorbate peroxidase; AT, 3aminotriazole; [Ca2+ ]cyt , cytosolic free calcium ion concentration; CAT, catalase; GPX, glutathione peroxidase; ICa channel, hyperpolarization-activated plasma membrane Ca2+ -permeable channel; MeJA, methyl jasmonate; MES, 2-(Nmorpholino)ethanesulfonic acid; ROS, reactive oxygen species; YC3.6, Yellow Cameleon 3.6. ∗ Corresponding author. Tel.: +81 86 434 1215; fax: +81 86 434 1249. E-mail address: [email protected] (I.C. Mori). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.03.006
cells, ultimately leading to stomatal closure (Allen et al., 2000; Pei et al., 2000; Mori et al., 2006; Munemasa et al., 2007). Exogenous application of catalase (CAT) inhibited MeJA- and ABA-induced stomatal closure and reduced the associated H2 O2 accumulation in guard cells (Zhang et al., 2001; Munemasa et al., 2007). CATs have been suggested to play a key role in the MeJA signaling as well as the ABA signaling. However, in vivo function of CATs in MeJA-induced stomatal closure remains to be clarified. In Arabidopsis, three CAT loci have been identified (Frugoli et al., 1996). Loss-of-function mutations of the guard cell-expressed CATs (CAT1 and CAT3) caused an increase of H2 O2 accumulation in guard cells and enhanced ABA sensitivity of stomata (Jannat et al., 2011b). Furthermore, it was suggested that the enhanced ABA-inducible ROS elevation potentiated stomatal closure, while the constitutive ROS accumulation by cat1 and cat3 mutations was not involved in stomatal closure (Jannat et al., 2011b). The enzyme activity and transcriptional level of CAT2 were the highest among CATs in the whole leaves (Mhamdi et al., 2010). Although CAT2 is not expressed in guard cells, it was suggested that it indirectly regulates guard cell ROS accumulation and ABA-induced stomatal closure in Arabidopsis (Jannat et al., 2011a). In this study, we examined the functions of CATs in MeJA-induced stomatal closure using these cat mutants and a CAT inhibitor. Materials and methods Plant materials We used two ecotypes of Arabidopsis thaliana (L.) Heynh., Wassilewskija (WS) and Columbia-0 (Col-0) as wild type. cat2
R. Jannat et al. / Journal of Plant Physiology 169 (2012) 1012–1016
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Fig. 1. Methyl jasmonate (MeJA)-induced stomatal closure and ROS production in guard cells. (A and B) Stomatal closure in Col-0, cat2, WS, cat3-1 and cat1 cat3. Each datum was obtained from 60 stomatal aperture measurements. (C and D) H2 O2 production. The vertical scale represents percentage of dichlorofluoresein (DCF) fluorescence intensity when normalized to wild type control. Each datum was obtained from 50 guard cells. Figures having same letters do not show significant difference at 5% level (one-way ANOVA with Dunnett’s post hoc test). Error bars represent standard deviation.
(Salk 076998) and Yellow Cameleon 3.6 (YC3.6)-expressing transformants were of Col-0 accession (Jannat et al., 2011a). cat3-1 T-DNA mutant and cat1 cat3 double deletion mutant were isolated from WS background as described previously (Jannat et al., 2011b). Plants were grown as reported previously (Jannat et al., 2011b). Measurement of stomatal aperture and H2 O2 Stomatal aperture width was determined according to Murata et al. (2001), with slight modification, in which ABA was replaced with 10 M MeJA. H2 O2 accumulation in guard cells was examined with 2 ,7 -dichlorodihydrofluorescein diacetate according to Jannat et al. (2011b). Measurement of [Ca2+ ]cyt oscillation in guard cells YC3.6-expressing cat2 plants were generated by crossing cat2 and YC3.6-expressing Col-0. [Ca2+ ]cyt oscillation in guard cells was examined as described by Hossain et al. (2011). In brief, the abaxial side of an excised leaf was gently attached to a glass slide using
Hollister medical adhesive. The adaxial epidermis and mesophyll tissues were then removed carefully with a razor blade to retain the intact abaxial epidermis on the slide. The epidermal specimens were incubated in a solution containing 5 mM KCl, 50 M CaCl2 and 10 mM MES-Tris (pH 6.15) under light for 2 h at 22 ◦ C to promote stomatal opening. Turgid guard cells (those not damaged by the razor blade) were considered for the measurement of [Ca2+ ]cyt oscillations. The specimen on the slide was exposed to MeJA by replacing the solutions with a peristaltic pump. Dualemission ratio imaging of YC3.6 was carried out as described by Jannat et al. (2011a). Results Effects of the cat mutations on MeJA-induced stomatal closure and ROS accumulation To elucidate the functional importance of CAT genes in the MeJA response of guard cells, we examined MeJA-induced stomatal closure and H2 O2 production in cat mutants (Fig. 1). It has
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Fig. 2. MeJA-induced stomatal closure and ROS production in plants pre-treated with the CAT inhibitor, 3-aminotriazole (AT). (A and B) Stomatal closure. Excised rosette leaves were treated with 20 mM AT for 30 min, followed by 10 M MeJA treatment for 2 h. Each datum was obtained from 60 stomatal aperture measurements. (C and D) H2 O2 production. Excised leaves were pretreated with 20 mM AT for 30 min, followed by 10 M MeJA treatment. The vertical scale represents the percentage of DCF fluorescent intensity when normalized to the control. Each datum was obtained from 50 guard cells. Figures having same letters do not show significant difference at 5% level (one-way ANOVA with Dunnett’s post hoc test). Error bars represent standard deviation.
been reported that CAT activity of these mutants was remarkably reduced (Jannat et al., 2011a,b). In the absence of MeJA, no difference was observed in stomatal aperture widths between the wild type and each cat mutant (Fig. 1A and B). As expected, 10 M MeJA induced stomatal closure in the wild type (p < 0.01, Col-0; p < 0.01, WS) and cat mutants (p < 0.01, cat2; p < 0.01, cat3-1; p < 0.01, cat1 cat3). The closure was potentiated by the mutations when compared with each corresponding wild type (p < 0.05 for cat2, cat3-1 and cat1 cat3). We next examined H2 O2 accumulation in guard cells (Fig. 1C and D). In the absence of MeJA, H2 O2 levels in guard cells of cat2 (p < 0.01), cat3-1 (p < 0.01) and cat1 cat3 (p < 0.01) were higher than that of the wild type. As reported by Islam et al. (2010) and Munemasa et al. (2011), MeJA increased H2 O2 levels in the wild type (p < 0.01, Col-0; p < 0.01, WS). The elevation of H2 O2 was also observed in mutant guard cells (cat2, p < 0.05; cat3-1, p < 0.01; cat1 cat3, p < 0.01). The elevated levels were significantly higher compared to the wild type (cat2, p < 0.05; cat3-1, p < 0.05; cat1 cat3, p < 0.05). These results closely mimic the responses to ABA (Jannat et al., 2011a,b). By analogy, we postulated that MeJA-induced
stomatal closure requires not just an elevation, but a specific inducible H2 O2 elevation; and MeJA and ABA signaling share the same CATs to down-regulate ROS accumulation (Jannat et al., 2011a,b and references therein).
Effects of the catalase inhibitor, 3-aminotriazole (AT), on MeJA-induced stomatal closure To further examine the involvement of CATs in MeJA-induced stomatal closure, we employed a pharmacological as well as a molecular genetics approach, viz. that the effect of AT was assessed (Fig. 2). AT treatment at the concentration that abolishes CAT activities (Jannat et al., 2011b), did not affect the stomatal aperture width of the wild type and mutants in the absence of MeJA. In the wild type, MeJA-induced stomatal closure was potentiated by AT (p < 0.01, Col-0; p < 0.01, WS) and reached a level similar to that in cat mutants. As for H2 O2 production, AT significantly increased the intracellular level in MeJA-untreated (p < 0.01, Col-0; p < 0.01, WS) and -treated (p < 0.01, Col-0; p < 0.01, WS) guard cells. The obtained
R. Jannat et al. / Journal of Plant Physiology 169 (2012) 1012–1016
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Fig. 3. MeJA-induced [Ca2+ ]cyt oscillations. (A, B, D, and E) [Ca2+ ]cyt oscillation induced by 10 M MeJA in wild type in the absence and presence of AT, and cat2 in the absence and presence of AT. (C and F) Stack column representation of MeJA-induced [Ca2+ ]cyt oscillation frequency (%) in wild type (n = 28) and cat2 guard cells (n = 28), and AT-untreated (n = 24) and -treated (n = 22) wild-type guard cells. Ca2+ oscillation was monitored for 60 min.
results were in remarkable agreement with cat mutants (Fig. 2C and D) and closely resemble the ABA responses (Jannat et al., 2011a,b). MeJA-induced [Ca2+ ]cyt oscillations in cat2 and AT-treated guard cells We examined the effects of cat2 mutation and AT treatment on MeJA-elicited [Ca2+ ]cyt oscillation, which is one of the hallmarks of MeJA signaling events in guard cells (Munemasa et al., 2007, 2011; Islam et al., 2010). MeJA treatment elicited one or more [Ca2+ ]cyt transient elevations (hereafter, [Ca2+ ]cyt oscillation) in the wild-type, cat2 and AT-treated guard cells (Fig. 3). No significant differences in oscillation frequency were detected between the mutant and wild type (2 test, ˛ = 0.05). In the absence of MeJA, [Ca2+ ]cyt oscillation was not detected in cat2 guard cells (data not shown), while the H2 O2 level was constitutively elevated (Fig. 1C). This indicates that the constitutive ROS elevation in the CAT mutant does not evoke the [Ca2+ ]cyt oscillation, while the MeJA-inducible ROS elevation seems to be closely involved and this resembles the ABA-inducible ROS elevation (Jannat et al., 2011a). Discussion Spatiotemporal and chemical species speciation of ROS message in guard cells MeJA induces stomatal closure accompanied by ROS production in guard cells similar to the way that ABA induces stomatal
closure (Pei et al., 2000; Kwak et al., 2003; Suhita et al., 2004; Munemasa et al., 2007, 2011). Diminution of CAT activity with the genetic mutations and the inhibitor treatment resulted in a constitutive H2 O2 accumulation in guard cells even in the absence of MeJA. However, the constitutive accumulation did not reduce the width of the stomatal aperture, nor elicit any [Ca2+ ]cyt oscillation. On the other hand, the reduction of CAT activity potentiated MeJAinduced stomatal closure and H2 O2 elevation. This genetic and pharmacological evidence suggests the involvement of CATs in regulating endogenous H2 O2 in guard cells. Furthermore the evidence also distinguishes the functions of constitutively accumulated and stimulus-induced H2 O2 . This may imply that spatiotemporal specific ROS signatures play a crucial role in signal discrimination in plant cells. Glutathione peroxidases (GPXs) reduce H2 O2 and organic hydroperoxide using the redox potential of thioredoxin in plants (Iqbal et al., 2006; Miao et al., 2006). The Arabidopsis GPX, AtGPX3, plays dual roles: it acts as a general hydroperoxide scavenger as well as relays H2 O2 signals in the ABA reaction (Miao et al., 2006). Ascorbate peroxidase 1 (APX1) is involved in the reaction of stomata to the light–dark transition, but not to ABA (Pnueli et al., 2003). APX1 mainly decomposes superoxide anion radicals and H2 O2 in cytosol (Davletova et al., 2005). The difference in substrate preference and subcellular localization of CAT, APX and GPX may be one of the reasons for the phenotypical differences among the mutants. Collectively, we hypothesize the roles of specific ROS as a messenger in guard cell signaling in a spatial–temporal manner.
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In a previous study, we showed that CAT2 functions in the guard cell ABA signaling, although it is not apparently expressed in guard cells (Jannat et al., 2011a). The higher H2 O2 accumulation in cat2 guard cells may be attributed to the supply of H2 O2 from peripheral cells, such as epidermal pavement cells and mesophyll cells, because H2 O2 is highly diffusible and permeable to biomembranes. Therefore it is conceivable that the stomatal closure-specific ROS message in guard cells is not a simple inward flow of H2 O2 across the plasma membrane. If this were the case, the H2 O2 influx from peripheral cells in cat2 would mimic the stomata-closing ROS message. Thus, we would like to propose that the logical addition of ROS signal messages and other signal messages drive the stomataclosing machinery. This hypothesis is intuitively acceptable, since a guard cell needs to integrate an array of environmental signals to control its aperture size. ROS signal and [Ca2+ ]cyt oscillation plasma membrane Ca2+ Hyperpolarization-activated permeable (ICa ) channels function in ABA- and MeJA-induced stomatal closure in Arabidopsis (Pei et al., 2000; Kwak et al., 2003; Mori et al., 2006; Munemasa et al., 2007). Application of H2 O2 to guard cells activates ICa channels and stimulates [Ca2+ ]cyt oscillation (Pei et al., 2000; Kwak et al., 2003), suggesting a close link between the generation of ROS and the initiation of the oscillation (Mori and Schroeder, 2004). In the ABA response of guard cells, the induction of [Ca2+ ]cyt oscillation was up-regulated when CAT activity was reduced (Jannat et al., 2011a,b). In this study, up-regulation of the oscillation was not clearly shown when MeJA was challenged (Fig. 3). This may be attributed to the technical limitations of the quantitative analysis of the frequency of oscillations. It is interesting to note that the constitutive H2 O2 accumulation did not cause the initiation of [Ca2+ ]cyt oscillation alongside stomatal closure. This indicates that the specific up-down patterns of intracellular H2 O2 level, rather than a simple elevation, function to transduce the signals. Similar concepts have been proposed in Ca2+ signaling. The Ca2+ plateau did not induce stomatal closure, and a specific signature of Ca2+ oscillation pattern is a prerequisite for stomatal closure (Allen et al., 2000; Young et al., 2006; Siegel et al., 2009). We hypothesize that the spatiotemporal specific ROS elevation patterns (ROS signature) function in ABA- and MeJA-induced stomatal closure in a harmonized way with the Ca2+ signature. Recently, it has been reported that Ca2+ -dependent peroxisomal H2 O2 catabolism is modulated by CAT3 in Arabidopsis guard cells (Costa et al., 2010), providing evidence for the close interaction between Ca2+ and ROS. NAD(P)H oxidase generates superoxide anion radicals extracellularly. On the other hand, extracellular application of membrane impermeable CAT, which does not decompose superoxide anion radicals, inhibited ABA- and MeJA-induced stomatal closure (Zhang et al., 2001; Munemasa et al., 2007). At a first glance, these facts seem paradoxical. However, this may be well explained by the model proposed by the previous study (Jannat et al., 2011b), where the generated superoxide anion radicals in apoplasts is converted to H2 O2 by superoxide dismutase in the extracellular space, and the generated H2 O2 moves into the cell resulting in the activation of signal transduction cascades.
In summary, the present study provides evidence that the CATs genes function in MeJA signal transduction by down-regulating the intracellular H2 O2 accumulation, which could, in turn, influence the subsequent signal transduction events. MeJA-induced stomatal closure requires inducible H2 O2 production. CATs commonly function to down-regulate ROS during ABA- and MeJA-induced stomatal closure. Acknowledgements This research was supported in part by Grant-in-Aid for Young Scientists and Grants for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Nissan Science Foundation, the Ohara Foundation for Agricultural Research and Special Account Budget for Education and Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Allen GJ, Chu SP, Schumacher K, Shimazaki CT, Vafeados D, Kemper A, et al. Science 2000;289:2338–42. Berger S, Bell E, Mullet JE. Plant Physiol 1996;111:525–31. Browse J. Annu Rev Plant Biol 2009;60:183–205. Costa A, Drago I, Behera S, Zottini M, Pizzo P, Schroeder JI, et al. Plant J 2010;62:760–72. Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, Coutu J, et al. Plant Cell 2005;17:268–81. Frugoli JA, Zhong HH, Nuccio ML, McCourt P, McPeek MA, Thomas TL, et al. Plant Physiol 1996;112:327–36. Gehring CA, Irving HR, McConchie R, Parish RW. Ann Bot 1997;80:485–9. Hetherington AM, Woodward FJ. Nature 2003;424:901–8. Hossain MA, Munemasa S, Uraji M, Nakammura Y, Mori IC, Murata Y. Plant Physiol 2011;156:430–8. Iqbal A, Yabuta Y, Takeda T, Nakano Y, Shigeoka S. FEBS J 2006;273:5589–97. Irving HR, Gehring CA, Parish RW. Proc Natl Acad Sci USA 1992;89:1790–4. Islam MM, Hossain MA, Jannat R, Munemasa S, Nakamura Y, Mori IC, et al. Plant Cell Physiol 2010;51:1721–30. Jannat R, Uraji M, Morofuji M, Hossain MA, Islam MM, Nakamura Y, et al. Biosci Biotechnol Biochem 2011a;75:2034–6. Jannat R, Uraji M, Morofuji M, Islam MM, Bloom RE, Nakamura Y, et al. J Plant Physiol 2011b;168:1919–26. Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, et al. EMBO J 2003;22:2623–33. Liu X, Zhang SQ, Lou CH, Yu FY. Acta Bot Sin 2002;44:164–7. Mhamdi A, Queval G, Chaouch S, Vanderauwera S, Breusegem FV, Noctor G. J Exp Bot 2010;61:4197–220. Miao Y, Lv D, Wang P, Wang X-C, Chen J, Miao C, et al. Plant Cell 2006;18:2749–66. Mori IC, Murata Y, Yang Y, Munemasa S, Wang Y-F, Andreoli S, et al. PLoS Biol 2006;4:1749–62. Mori IC, Schroeder JI. Plant Physiol 2004;135:702–8. Munemasa S, Oda K, Watanabe-Sugimoto M, Nakamura Y, Shimoishi Y, Murata Y. Plant Physiol 2007;143:1398–407. Munemasa S, Hossain MA, Nakamura Y, Mori IC, Murata Y. Plant Physiol 2011;155:553–61. Murata Y, Pei ZM, Mori IC, Schroeder JI. Plant Cell 2001;13:2513–23. Pei ZM, Murata Y, Benning G, Thomine S, Kl¨vsener B, Allen GJ, et al. Nature 2000;406:731–4. Pnueli L, Liang H, Rozenberg M, Mittler R. Plant J 2003;34:187–203. Saito N, Munemasa S, Nakamura Y, Shimoishi Y, Mori IC, Murata Y. Plant Cell Physiol 2008;49:1396–401. Schroeder JI, Allen GJ, Hugouvieux V, Kwak JM, Waner D. Ann Rev Plant Physiol Plant Mol Biol 2001;52:627–58. Siegel RS, Xue S, Murata Y, Yang Y, Nishimura N, Wang A, et al. Plant J 2009;59:207–20. Suhita D, Agepati S, Raghavendra AS, Kwak JM, Vavasseur A. Plant Physiol 2004;134:1536–45. Young JJ, Mehta S, Israelsson M, Godoski J, Grill E, Schroeder JI. Proc Natl Acad Sci USA 2006;103:7506–11. Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, Song CP. Plant Physiol 2001;126:1438–48.
Contents lists available at SciVerse ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.de/jplph
Short communication
Catalases negatively regulate methyl jasmonate signaling in guard cells Rayhanur Jannata , Misugi Urajia , Mohammad Anowar Hossaina , Mohammad Muzahidul Islama , Yoshimasa Nakamuraa , Izumi C. Morib,∗ , Yoshiyuki Murataa a b
Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan Institute of Plant Science and Resources, Okayama University, 2-20-1, Kurashiki 710-8530, Japan
a r t i c l e
i n f o
Article history: Received 6 December 2011 Received in revised form 29 March 2012 Accepted 30 March 2012 Keywords: Catalase Guard cells Hydrogen peroxide Methyl jasmonate
a b s t r a c t Methyl jasmonate (MeJA)-induced stomatal closure is accompanied by the accumulation of hydrogen peroxide (H2 O2 ) in guard cells. In this study, we investigated the roles of catalases (CATs) in MeJA-induced stomatal closure using cat mutants cat2, cat3-1 and cat1 cat3, and the CAT inhibitor, 3-aminotriazole (AT). When assessed with 2 ,7 -dichlorodihydrofluorescein, the reduction of catalase activity by means of mutations and the inhibitor accumulated higher basal levels of H2 O2 in guard cells whereas they did not affect stomatal aperture in the absence of MeJA. In contrast, the cat mutations and the treatment with AT potentiated MeJA-induced stomatal closure and MeJA-induced H2 O2 production. These results indicate that CATs negatively regulate H2 O2 accumulation in guard cells and suggest that inducible H2 O2 production rather than constitutive elevation modulates stomatal apertures in Arabidopsis. © 2012 Elsevier GmbH. All rights reserved.
Introduction The stomatal apparatus, which consists of a pair of guard cells in the epidermis of above-ground parts of higher plants, responds to various external and internal stimuli (Schroeder et al., 2001; Hetherington and Woodward, 2003). Stomatal apertures close when exposed to the volatile phytohormone, methyl jasmonate (MeJA), which regulates various physiological processes and reacts to wounding and pathogen attack (Berger et al., 1996; Browse, 2009) by limiting the invasion of pathogens through the pores (Irving et al., 1992; Gehring et al., 1997; Liu et al., 2002; Suhita et al., 2004; Munemasa et al., 2007; Saito et al., 2008; Islam et al., 2010). Hydrogen peroxide (H2 O2 ) is one of the major reactive oxygen species (ROS) and acts as a second messenger in MeJA and abscisic acid (ABA) signal transduction in guard cells. Genetic and pharmacological studies have demonstrated that the ROS production is mediated by NAD(P)H oxidases (Kwak et al., 2003; Suhita et al., 2004). The generated ROS activates plasma membrane Ca2+ permeable channels (ICa channels), which in turn elicit cytosolic free calcium ion concentration ([Ca2+ ]cyt ) elevation/oscillation in guard
Abbreviations: ABA, abscisic acid; APX, ascorbate peroxidase; AT, 3aminotriazole; [Ca2+ ]cyt , cytosolic free calcium ion concentration; CAT, catalase; GPX, glutathione peroxidase; ICa channel, hyperpolarization-activated plasma membrane Ca2+ -permeable channel; MeJA, methyl jasmonate; MES, 2-(Nmorpholino)ethanesulfonic acid; ROS, reactive oxygen species; YC3.6, Yellow Cameleon 3.6. ∗ Corresponding author. Tel.: +81 86 434 1215; fax: +81 86 434 1249. E-mail address: [email protected] (I.C. Mori). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.03.006
cells, ultimately leading to stomatal closure (Allen et al., 2000; Pei et al., 2000; Mori et al., 2006; Munemasa et al., 2007). Exogenous application of catalase (CAT) inhibited MeJA- and ABA-induced stomatal closure and reduced the associated H2 O2 accumulation in guard cells (Zhang et al., 2001; Munemasa et al., 2007). CATs have been suggested to play a key role in the MeJA signaling as well as the ABA signaling. However, in vivo function of CATs in MeJA-induced stomatal closure remains to be clarified. In Arabidopsis, three CAT loci have been identified (Frugoli et al., 1996). Loss-of-function mutations of the guard cell-expressed CATs (CAT1 and CAT3) caused an increase of H2 O2 accumulation in guard cells and enhanced ABA sensitivity of stomata (Jannat et al., 2011b). Furthermore, it was suggested that the enhanced ABA-inducible ROS elevation potentiated stomatal closure, while the constitutive ROS accumulation by cat1 and cat3 mutations was not involved in stomatal closure (Jannat et al., 2011b). The enzyme activity and transcriptional level of CAT2 were the highest among CATs in the whole leaves (Mhamdi et al., 2010). Although CAT2 is not expressed in guard cells, it was suggested that it indirectly regulates guard cell ROS accumulation and ABA-induced stomatal closure in Arabidopsis (Jannat et al., 2011a). In this study, we examined the functions of CATs in MeJA-induced stomatal closure using these cat mutants and a CAT inhibitor. Materials and methods Plant materials We used two ecotypes of Arabidopsis thaliana (L.) Heynh., Wassilewskija (WS) and Columbia-0 (Col-0) as wild type. cat2
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Fig. 1. Methyl jasmonate (MeJA)-induced stomatal closure and ROS production in guard cells. (A and B) Stomatal closure in Col-0, cat2, WS, cat3-1 and cat1 cat3. Each datum was obtained from 60 stomatal aperture measurements. (C and D) H2 O2 production. The vertical scale represents percentage of dichlorofluoresein (DCF) fluorescence intensity when normalized to wild type control. Each datum was obtained from 50 guard cells. Figures having same letters do not show significant difference at 5% level (one-way ANOVA with Dunnett’s post hoc test). Error bars represent standard deviation.
(Salk 076998) and Yellow Cameleon 3.6 (YC3.6)-expressing transformants were of Col-0 accession (Jannat et al., 2011a). cat3-1 T-DNA mutant and cat1 cat3 double deletion mutant were isolated from WS background as described previously (Jannat et al., 2011b). Plants were grown as reported previously (Jannat et al., 2011b). Measurement of stomatal aperture and H2 O2 Stomatal aperture width was determined according to Murata et al. (2001), with slight modification, in which ABA was replaced with 10 M MeJA. H2 O2 accumulation in guard cells was examined with 2 ,7 -dichlorodihydrofluorescein diacetate according to Jannat et al. (2011b). Measurement of [Ca2+ ]cyt oscillation in guard cells YC3.6-expressing cat2 plants were generated by crossing cat2 and YC3.6-expressing Col-0. [Ca2+ ]cyt oscillation in guard cells was examined as described by Hossain et al. (2011). In brief, the abaxial side of an excised leaf was gently attached to a glass slide using
Hollister medical adhesive. The adaxial epidermis and mesophyll tissues were then removed carefully with a razor blade to retain the intact abaxial epidermis on the slide. The epidermal specimens were incubated in a solution containing 5 mM KCl, 50 M CaCl2 and 10 mM MES-Tris (pH 6.15) under light for 2 h at 22 ◦ C to promote stomatal opening. Turgid guard cells (those not damaged by the razor blade) were considered for the measurement of [Ca2+ ]cyt oscillations. The specimen on the slide was exposed to MeJA by replacing the solutions with a peristaltic pump. Dualemission ratio imaging of YC3.6 was carried out as described by Jannat et al. (2011a). Results Effects of the cat mutations on MeJA-induced stomatal closure and ROS accumulation To elucidate the functional importance of CAT genes in the MeJA response of guard cells, we examined MeJA-induced stomatal closure and H2 O2 production in cat mutants (Fig. 1). It has
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Fig. 2. MeJA-induced stomatal closure and ROS production in plants pre-treated with the CAT inhibitor, 3-aminotriazole (AT). (A and B) Stomatal closure. Excised rosette leaves were treated with 20 mM AT for 30 min, followed by 10 M MeJA treatment for 2 h. Each datum was obtained from 60 stomatal aperture measurements. (C and D) H2 O2 production. Excised leaves were pretreated with 20 mM AT for 30 min, followed by 10 M MeJA treatment. The vertical scale represents the percentage of DCF fluorescent intensity when normalized to the control. Each datum was obtained from 50 guard cells. Figures having same letters do not show significant difference at 5% level (one-way ANOVA with Dunnett’s post hoc test). Error bars represent standard deviation.
been reported that CAT activity of these mutants was remarkably reduced (Jannat et al., 2011a,b). In the absence of MeJA, no difference was observed in stomatal aperture widths between the wild type and each cat mutant (Fig. 1A and B). As expected, 10 M MeJA induced stomatal closure in the wild type (p < 0.01, Col-0; p < 0.01, WS) and cat mutants (p < 0.01, cat2; p < 0.01, cat3-1; p < 0.01, cat1 cat3). The closure was potentiated by the mutations when compared with each corresponding wild type (p < 0.05 for cat2, cat3-1 and cat1 cat3). We next examined H2 O2 accumulation in guard cells (Fig. 1C and D). In the absence of MeJA, H2 O2 levels in guard cells of cat2 (p < 0.01), cat3-1 (p < 0.01) and cat1 cat3 (p < 0.01) were higher than that of the wild type. As reported by Islam et al. (2010) and Munemasa et al. (2011), MeJA increased H2 O2 levels in the wild type (p < 0.01, Col-0; p < 0.01, WS). The elevation of H2 O2 was also observed in mutant guard cells (cat2, p < 0.05; cat3-1, p < 0.01; cat1 cat3, p < 0.01). The elevated levels were significantly higher compared to the wild type (cat2, p < 0.05; cat3-1, p < 0.05; cat1 cat3, p < 0.05). These results closely mimic the responses to ABA (Jannat et al., 2011a,b). By analogy, we postulated that MeJA-induced
stomatal closure requires not just an elevation, but a specific inducible H2 O2 elevation; and MeJA and ABA signaling share the same CATs to down-regulate ROS accumulation (Jannat et al., 2011a,b and references therein).
Effects of the catalase inhibitor, 3-aminotriazole (AT), on MeJA-induced stomatal closure To further examine the involvement of CATs in MeJA-induced stomatal closure, we employed a pharmacological as well as a molecular genetics approach, viz. that the effect of AT was assessed (Fig. 2). AT treatment at the concentration that abolishes CAT activities (Jannat et al., 2011b), did not affect the stomatal aperture width of the wild type and mutants in the absence of MeJA. In the wild type, MeJA-induced stomatal closure was potentiated by AT (p < 0.01, Col-0; p < 0.01, WS) and reached a level similar to that in cat mutants. As for H2 O2 production, AT significantly increased the intracellular level in MeJA-untreated (p < 0.01, Col-0; p < 0.01, WS) and -treated (p < 0.01, Col-0; p < 0.01, WS) guard cells. The obtained
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Fig. 3. MeJA-induced [Ca2+ ]cyt oscillations. (A, B, D, and E) [Ca2+ ]cyt oscillation induced by 10 M MeJA in wild type in the absence and presence of AT, and cat2 in the absence and presence of AT. (C and F) Stack column representation of MeJA-induced [Ca2+ ]cyt oscillation frequency (%) in wild type (n = 28) and cat2 guard cells (n = 28), and AT-untreated (n = 24) and -treated (n = 22) wild-type guard cells. Ca2+ oscillation was monitored for 60 min.
results were in remarkable agreement with cat mutants (Fig. 2C and D) and closely resemble the ABA responses (Jannat et al., 2011a,b). MeJA-induced [Ca2+ ]cyt oscillations in cat2 and AT-treated guard cells We examined the effects of cat2 mutation and AT treatment on MeJA-elicited [Ca2+ ]cyt oscillation, which is one of the hallmarks of MeJA signaling events in guard cells (Munemasa et al., 2007, 2011; Islam et al., 2010). MeJA treatment elicited one or more [Ca2+ ]cyt transient elevations (hereafter, [Ca2+ ]cyt oscillation) in the wild-type, cat2 and AT-treated guard cells (Fig. 3). No significant differences in oscillation frequency were detected between the mutant and wild type (2 test, ˛ = 0.05). In the absence of MeJA, [Ca2+ ]cyt oscillation was not detected in cat2 guard cells (data not shown), while the H2 O2 level was constitutively elevated (Fig. 1C). This indicates that the constitutive ROS elevation in the CAT mutant does not evoke the [Ca2+ ]cyt oscillation, while the MeJA-inducible ROS elevation seems to be closely involved and this resembles the ABA-inducible ROS elevation (Jannat et al., 2011a). Discussion Spatiotemporal and chemical species speciation of ROS message in guard cells MeJA induces stomatal closure accompanied by ROS production in guard cells similar to the way that ABA induces stomatal
closure (Pei et al., 2000; Kwak et al., 2003; Suhita et al., 2004; Munemasa et al., 2007, 2011). Diminution of CAT activity with the genetic mutations and the inhibitor treatment resulted in a constitutive H2 O2 accumulation in guard cells even in the absence of MeJA. However, the constitutive accumulation did not reduce the width of the stomatal aperture, nor elicit any [Ca2+ ]cyt oscillation. On the other hand, the reduction of CAT activity potentiated MeJAinduced stomatal closure and H2 O2 elevation. This genetic and pharmacological evidence suggests the involvement of CATs in regulating endogenous H2 O2 in guard cells. Furthermore the evidence also distinguishes the functions of constitutively accumulated and stimulus-induced H2 O2 . This may imply that spatiotemporal specific ROS signatures play a crucial role in signal discrimination in plant cells. Glutathione peroxidases (GPXs) reduce H2 O2 and organic hydroperoxide using the redox potential of thioredoxin in plants (Iqbal et al., 2006; Miao et al., 2006). The Arabidopsis GPX, AtGPX3, plays dual roles: it acts as a general hydroperoxide scavenger as well as relays H2 O2 signals in the ABA reaction (Miao et al., 2006). Ascorbate peroxidase 1 (APX1) is involved in the reaction of stomata to the light–dark transition, but not to ABA (Pnueli et al., 2003). APX1 mainly decomposes superoxide anion radicals and H2 O2 in cytosol (Davletova et al., 2005). The difference in substrate preference and subcellular localization of CAT, APX and GPX may be one of the reasons for the phenotypical differences among the mutants. Collectively, we hypothesize the roles of specific ROS as a messenger in guard cell signaling in a spatial–temporal manner.
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In a previous study, we showed that CAT2 functions in the guard cell ABA signaling, although it is not apparently expressed in guard cells (Jannat et al., 2011a). The higher H2 O2 accumulation in cat2 guard cells may be attributed to the supply of H2 O2 from peripheral cells, such as epidermal pavement cells and mesophyll cells, because H2 O2 is highly diffusible and permeable to biomembranes. Therefore it is conceivable that the stomatal closure-specific ROS message in guard cells is not a simple inward flow of H2 O2 across the plasma membrane. If this were the case, the H2 O2 influx from peripheral cells in cat2 would mimic the stomata-closing ROS message. Thus, we would like to propose that the logical addition of ROS signal messages and other signal messages drive the stomataclosing machinery. This hypothesis is intuitively acceptable, since a guard cell needs to integrate an array of environmental signals to control its aperture size. ROS signal and [Ca2+ ]cyt oscillation plasma membrane Ca2+ Hyperpolarization-activated permeable (ICa ) channels function in ABA- and MeJA-induced stomatal closure in Arabidopsis (Pei et al., 2000; Kwak et al., 2003; Mori et al., 2006; Munemasa et al., 2007). Application of H2 O2 to guard cells activates ICa channels and stimulates [Ca2+ ]cyt oscillation (Pei et al., 2000; Kwak et al., 2003), suggesting a close link between the generation of ROS and the initiation of the oscillation (Mori and Schroeder, 2004). In the ABA response of guard cells, the induction of [Ca2+ ]cyt oscillation was up-regulated when CAT activity was reduced (Jannat et al., 2011a,b). In this study, up-regulation of the oscillation was not clearly shown when MeJA was challenged (Fig. 3). This may be attributed to the technical limitations of the quantitative analysis of the frequency of oscillations. It is interesting to note that the constitutive H2 O2 accumulation did not cause the initiation of [Ca2+ ]cyt oscillation alongside stomatal closure. This indicates that the specific up-down patterns of intracellular H2 O2 level, rather than a simple elevation, function to transduce the signals. Similar concepts have been proposed in Ca2+ signaling. The Ca2+ plateau did not induce stomatal closure, and a specific signature of Ca2+ oscillation pattern is a prerequisite for stomatal closure (Allen et al., 2000; Young et al., 2006; Siegel et al., 2009). We hypothesize that the spatiotemporal specific ROS elevation patterns (ROS signature) function in ABA- and MeJA-induced stomatal closure in a harmonized way with the Ca2+ signature. Recently, it has been reported that Ca2+ -dependent peroxisomal H2 O2 catabolism is modulated by CAT3 in Arabidopsis guard cells (Costa et al., 2010), providing evidence for the close interaction between Ca2+ and ROS. NAD(P)H oxidase generates superoxide anion radicals extracellularly. On the other hand, extracellular application of membrane impermeable CAT, which does not decompose superoxide anion radicals, inhibited ABA- and MeJA-induced stomatal closure (Zhang et al., 2001; Munemasa et al., 2007). At a first glance, these facts seem paradoxical. However, this may be well explained by the model proposed by the previous study (Jannat et al., 2011b), where the generated superoxide anion radicals in apoplasts is converted to H2 O2 by superoxide dismutase in the extracellular space, and the generated H2 O2 moves into the cell resulting in the activation of signal transduction cascades.
In summary, the present study provides evidence that the CATs genes function in MeJA signal transduction by down-regulating the intracellular H2 O2 accumulation, which could, in turn, influence the subsequent signal transduction events. MeJA-induced stomatal closure requires inducible H2 O2 production. CATs commonly function to down-regulate ROS during ABA- and MeJA-induced stomatal closure. Acknowledgements This research was supported in part by Grant-in-Aid for Young Scientists and Grants for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Nissan Science Foundation, the Ohara Foundation for Agricultural Research and Special Account Budget for Education and Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References Allen GJ, Chu SP, Schumacher K, Shimazaki CT, Vafeados D, Kemper A, et al. Science 2000;289:2338–42. Berger S, Bell E, Mullet JE. Plant Physiol 1996;111:525–31. Browse J. Annu Rev Plant Biol 2009;60:183–205. Costa A, Drago I, Behera S, Zottini M, Pizzo P, Schroeder JI, et al. Plant J 2010;62:760–72. Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, Coutu J, et al. Plant Cell 2005;17:268–81. Frugoli JA, Zhong HH, Nuccio ML, McCourt P, McPeek MA, Thomas TL, et al. Plant Physiol 1996;112:327–36. Gehring CA, Irving HR, McConchie R, Parish RW. Ann Bot 1997;80:485–9. Hetherington AM, Woodward FJ. Nature 2003;424:901–8. Hossain MA, Munemasa S, Uraji M, Nakammura Y, Mori IC, Murata Y. Plant Physiol 2011;156:430–8. Iqbal A, Yabuta Y, Takeda T, Nakano Y, Shigeoka S. FEBS J 2006;273:5589–97. Irving HR, Gehring CA, Parish RW. Proc Natl Acad Sci USA 1992;89:1790–4. Islam MM, Hossain MA, Jannat R, Munemasa S, Nakamura Y, Mori IC, et al. Plant Cell Physiol 2010;51:1721–30. Jannat R, Uraji M, Morofuji M, Hossain MA, Islam MM, Nakamura Y, et al. Biosci Biotechnol Biochem 2011a;75:2034–6. Jannat R, Uraji M, Morofuji M, Islam MM, Bloom RE, Nakamura Y, et al. J Plant Physiol 2011b;168:1919–26. Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, et al. EMBO J 2003;22:2623–33. Liu X, Zhang SQ, Lou CH, Yu FY. Acta Bot Sin 2002;44:164–7. Mhamdi A, Queval G, Chaouch S, Vanderauwera S, Breusegem FV, Noctor G. J Exp Bot 2010;61:4197–220. Miao Y, Lv D, Wang P, Wang X-C, Chen J, Miao C, et al. Plant Cell 2006;18:2749–66. Mori IC, Murata Y, Yang Y, Munemasa S, Wang Y-F, Andreoli S, et al. PLoS Biol 2006;4:1749–62. Mori IC, Schroeder JI. Plant Physiol 2004;135:702–8. Munemasa S, Oda K, Watanabe-Sugimoto M, Nakamura Y, Shimoishi Y, Murata Y. Plant Physiol 2007;143:1398–407. Munemasa S, Hossain MA, Nakamura Y, Mori IC, Murata Y. Plant Physiol 2011;155:553–61. Murata Y, Pei ZM, Mori IC, Schroeder JI. Plant Cell 2001;13:2513–23. Pei ZM, Murata Y, Benning G, Thomine S, Kl¨vsener B, Allen GJ, et al. Nature 2000;406:731–4. Pnueli L, Liang H, Rozenberg M, Mittler R. Plant J 2003;34:187–203. Saito N, Munemasa S, Nakamura Y, Shimoishi Y, Mori IC, Murata Y. Plant Cell Physiol 2008;49:1396–401. Schroeder JI, Allen GJ, Hugouvieux V, Kwak JM, Waner D. Ann Rev Plant Physiol Plant Mol Biol 2001;52:627–58. Siegel RS, Xue S, Murata Y, Yang Y, Nishimura N, Wang A, et al. Plant J 2009;59:207–20. Suhita D, Agepati S, Raghavendra AS, Kwak JM, Vavasseur A. Plant Physiol 2004;134:1536–45. Young JJ, Mehta S, Israelsson M, Godoski J, Grill E, Schroeder JI. Proc Natl Acad Sci USA 2006;103:7506–11. Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, Song CP. Plant Physiol 2001;126:1438–48.