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Allyl isothiocyanate induces stomatal closure in Vicia faba Muhammad Abdus Sobahan, Nasima Akter, Eiji Okuma, Misugi Uraji, Wenxiu Ye, Izumi C. Mori, Yoshimasa Nakamura & Yoshiyuki Murata To cite this article: Muhammad Abdus Sobahan, Nasima Akter, Eiji Okuma, Misugi Uraji, Wenxiu Ye, Izumi C. Mori, Yoshimasa Nakamura & Yoshiyuki Murata (2015) Allyl isothiocyanate induces stomatal closure in Vicia faba, Bioscience, Biotechnology, and Biochemistry, 79:10, 1737-1742, DOI: 10.1080/09168451.2015.1045827 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1045827
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Date: 08 November 2015, At: 19:37
Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 10, 1737–1742
Allyl isothiocyanate induces stomatal closure in Vicia faba Muhammad Abdus Sobahan, Nasima Akter, Eiji Okuma, Misugi Uraji, Wenxiu Ye, Izumi C. Mori, Yoshimasa Nakamura and Yoshiyuki Murata* Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan Received February 5, 2015; accepted April 22, 2015
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http://dx.doi.org/10.1080/09168451.2015.1045827
Isothiocyanates are enzymatically produced from glucosinolates in plants, and allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis thaliana. In this study, we investigated stomatal responses to AITC in Vicia faba. AITC-induced stomatal closure accompanied by reactive oxygen species (ROS) and NO production, cytosolic alkalization and glutathione (GSH) depletion in V. faba. GSH monoethyl ester induced stomatal reopening and suppressed AITC-induced GSH depletion in guard cells. Exogenous catalase and a peroxidase inhibitor, salicylhydroxamic acid, inhibited AITC-induced stomatal closure, unlike an NAD(P)H oxidase inhibitor, diphenylene iodonium chloride. The peroxidase inhibitor also abolished the AITC-induced ROS production, NO production, and cytosolic alkalization. AITC-induced stomatal closure was suppressed by an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide, and an agent to acidify cytosol, butyrate. These results indicate that AITC-induced stomatal closure in V. faba as well as in A. thaliana and suggest that AITC signaling in guard cells is conserved in both plants. Key words:
guard cells; peroxidase; stomatal closure
signaling;
Isothiocyanates (ITCs) that have biocidal and repellent activities against a wide range of pests are volatile organic compounds formed by the hydrolysis catalyzed by myrosinases of glucosinolates mostly in Brassicaceae plants.1) We have demonstrated that AITC, one of major ITCs, induces stomatal closure in Arabidopsis thaliana.2) It is unknown whether AITC induces stomatal responses in other plant taxa. Reactive oxygen species (ROS) and NO function as important secondary messengers in stomatal closure induced by various stimuli in a variety of plants including A. thaliana.3–8) In A. thaliana guard cells, AITC-induced ROS accumulation is mediated by salicylhydroxamic acid (SHAM)-sensitive peroxidases and AITC-induced NO production is dependent on the ROS accumulation.9) Glutathione (GSH) is the most
abundant non-protein thiol compound in plants and functions as a negative regulator in abscisic acid- and methyl jasmonate-induced stomatal closure, which are accompanied by GSH depletion in A. thaliana guard cells.10–12) In A. thaliana guard cells, AITC also induced GSH depletion and increasing intracellular GSH suppressed AITC-induced stomatal closure.2) Moreover, cytosolic alkalization is an important signaling event in stomatal closure.13–16) However, signaling mechanisms involved in AITC-induced stomatal closure in other plant taxa than A. thaliana remain to be clarified. Vicia faba belongs to Fabaceae but not to sixteen families of dicotyledonous angiosperms in which glucosinolates were present17) and is well studied as a material for stomatal movement. Hence, we employed V. faba as a material to examine stomatal responses to AITC of plants that do not belong to Brassicales order.
Materials and methods Plant materials and growth conditions. Broad bean (Vicia faba L., cv. House Ryousai) plants were grown in plastic pots (15 × 18 × 14 cm) filled with 70% (v/v) vermiculite (Asahi-Kogyo, Okayama, Japan) and 30% (v/v) Kureha soil (Kureha Chemical, Tokyo, Japan) in a growth chamber (22 ± 2 °C, 80 μmol m−2 s−1 under a 16-h-light/8-h-dark regime). Measurement of stomatal aperture. Stomatal aperture measurements were performed as described previously.18) Measurement of GSH in guard cells. Contents of GSH in guard cells were examined using monochlorobimane as previously described.10) Measurement of ROS and NO production in guard cells. Production of ROS and NO in guard cells was evaluated using 2′,7′-dichlorodihydrofluorescein
*Corresponding author. Email: [email protected]; Current address of Sobahan MA: School of Agriculture and Rural Development, Bangladesh Open University, Gazipur, Bangladesh. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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diacetate (H2DCF-DA) (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 4,5-diaminofluorescein-2 diacetate (DAF-2DA), respectively.16)
(Fig. 1(D)). These results suggest that GSH negatively regulates AITC-induced stomatal closure in V. faba as well as in A. thaliana.2)
Measurement of pHcyt. Cytosolic pH (pHcyt) in guard cells was analyzed using 2′,7′-bis(carboxyethyl)5,6-carboxyfluorescein acetomethylester (BCECF-AM) as previously described.15)
ROS are involved in AITC-induced stomatal closure in V. faba In A. thaliana guard cells, SHAM-sensitive peroxidases mediate ROS production induced by stimuli such as AITC, salicylic acid and elicitors,2,9,18) while NAD (P)H oxidases mediate ROS production induced by stimuli such as abscisic acid and methyl jasmonate.6,21) To examine the involvement of ROS in AITC-induced stomatal closure in V. faba, we examined the effects of peroxidase inhibitors, SHAM and sodium azide (NaN3), the hydrogen peroxide scavenger, catalase (CAT), and the NAD(P)H oxidase inhibitor, diphenylene iodonium chloride (DPI). As shown in Fig. 2(A), SHAM (p < 10−4), NaN3 (p < 0.001), and CAT (p < 10−4) strongly inhibited AITC-induced stomatal closure. However, the inhibition by DPI was not observed (p = 0.63). Neither SHAM (p = 0.88), NaN3 (p = 0.23), CAT (p = 0.17), nor DPI (p = 0.40) affected stomatal aperture in V. faba untreated with AITC (Fig. 2(B)). As shown in Fig. 2(C), 50 μM AITC-induced ROS accumulation in guard cells (p < 0.002). AITC failed to increase ROS level in guard cells in the presence of 2 mM SHAM (p = 0.17), NaN3 (p = 0.10), and CAT (p = 0.89) (Fig. 2(C)). These results indicate that AITC-induced stomatal closure is accompanied by ROS production mediated by SHAM-sensitive peroxidases in V. faba.
Treatment with gaseous AITC. To examine the effects of gaseous AITC, the excised leaves (15 × 10 mm) were floated on the incubation medium in an air-tight transparent plastic jar (420 ml) for 2 h in the light (80 μmol m−2 s−1) to open stomata. The desired amount of AITC (density: 1.013 g ml−1) was dropped on another dish in the jar and was spontaneously vaporized in a few minutes, and then, the leaves were incubated for another 2 h. Note that all amount of AITC was vaporized within 5 min on the dish in the jar. After the incubation, stomatal apertures were measured. Statistical analysis. Mean values were compared using Student’s t-test. Differences were considered significant for p values < 0.05.
Results AITC-induced stomatal closure accompanied by GSH depletion in V. faba Application of 10, 50, and 100 μM AITC narrowed stomatal apertures by 22% (p < 0.01), 37% (p < 10−4), and 43% (p < 10−4), respectively (Fig. 1(A)). There is no significant difference in stomata between no treatment control and the solvent control (p = 0.23). Gaseous AITC also induced stomatal closure in a dosedependent manner (Fig. 1(B)), suggesting that V. faba perceives gaseous AITC as well as dissolved AITC even though it does not produce ITCs. High concentrations of ITCs are toxic to cells. We tested viability of guard cells using fluorescein diacetate and neutral red. Guard cells treated with 10, 50, and 100 μM AITC for 2 h were stained by both dyes (data not shown), suggesting that AITC induces stomatal closure without reducing viability in V. faba. GSH can enzymatically and non-enzymatically react with ITCs,19) and membrane-permeable GSH monoethyl ester (GSHmee) can increase GSH contents in A. thaliana guard cells.10,20) To investigate whether the AITC-induced stomatal closure is reversible and whether GSH is involved in the stomatal closure in V. faba, the following experiments were performed. The epidermal tissues were incubated in the incubation medium containing AITC for 30 min to induce stomatal closure, followed by the addition of GSHmee to the medium. Subsequently, stomatal aperture width was measured (Fig. 1(C)). GSHmee significantly induced stomatal reopening (Fig. 1(C)). These results suggest that stomata of V. faba can reversibly respond to AITC. AITC decreased GSH in guard cells, which was significantly suppressed by treatment with GSHmee
NO was involved in AITC-induced stomatal closure in V. faba AITC-induced stomatal closure was suppressed by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (p < 0.001), where cPTIO alone did not affect stomatal aperture (p = 0.81) (Fig. 3(A)). As shown in Fig. 3(B), 50 μM AITC-induced NO production (p < 0.007, black bar) in guard cells, which was cancelled by 2 mM SHAM (p = 0.26, gray bar). These results indicate that NO production occurs in the process of AITC-induced stomatal closure, and the NO production depends on the SHAM-sensitive peroxidases in V. faba. Cytosolic alkalization was involved in AITC-induced stomatal closure in V. faba The AITC-induced stomatal closure was suppressed by the cytosol-acidifying agent, sodium butyrate (p < 0.001), where sodium butyrate alone did not affect stomatal aperture (p = 0.47) (Fig. 4(A)). As shown in Fig. 4(B), 50 μM AITC increased fluorescent intensity of BCECF (p < 0.004), indicating that AITC induces cytosolic alkalization in guard cells. AITC did not elicit cytosolic alkalization in guard cells when treated with 2 mM SHAM (p = 0.53) (Fig. 4(B)), suggesting that AITC-induced cytosolic alkalization depends on ROS production mediated by SHAMsensitive peroxidases.
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Fig. 1. Stomatal response to AITC in Vicia faba. Notes: (A) AITC-induced stomatal closure in V. faba. The epidermal tissues from V. faba leaves were treated with 10, 50, and 100 μM AITC for 2 h (black bars). White bar, control; dotted bar, dimethyl sulfoxide (DMSO). Data are the means of three independent experiments (60 total stomata per bar). (B) Gaseous AITC induced stomatal closure. The excised leaves of V. faba were floated on the incubation medium in an air-tight transparent plastic jar (420 ml) with 1, 5, and 10 μmol liquid AITC (black bars). Note that all amount of AITC was vaporized within 5 min on the dish in the jar. White bar, non-treatment control. Data are the means of three independent experiments (60 total stomata per bar). (C) Reopen time course of stomata pretreated with AITC by GSHmee. The epidermal preparation of V. faba leaves pretreated with 50 μM AITC for 30 min was rinsed with distilled water to remove excess AITC and incubated in the incubation medium supplemented with 10 μM GSHmee (open circles), DMSO (open triangles), or control (closed triangles). Data are the means of three independent experiments (60 total stomata per point). (D) Effects of AITC and GSHmee on GSH content in V. faba guard cells. White bar, control; divot bar, DMSO; black bar, 50 μM AITC; gray bars, 50 μM AITC and 10 μM GSHmee. Abaxial epidermal leaf tissues pre-treated with 10 μM GSHmee for 30 min were treated with 50 μM AITC. Each datum was obtained from at least 60 guard cells (n = 3). Error bars represent standard errors. Asterisk (*) denotes significant difference from the solvent control (p < 0.05).
Discussion AITC induces stomatal closure in the crucifer plant, A. thaliana, which can produce ITCs.2,9) In V. faba, AITC also induced stomatal closure (Fig. 1). This result suggests that V. faba possess a potential signal pathway leading to stomatal closure in response to AITC and that AITC-induced stomatal closure is not unique to A. thaliana but is common to plants. To our knowledge, it is unclear whether V. faba can biosynthesize glucosinolates. Separately, isoflavone, which is prevalent in legumes, is found to exist in A. thaliana although A. thaliana does not possess the genetic machinery (annotation of genome) of isoflavone synthesis.22) Hence, we cannot exclude the possibility that glucosinolates are biosynthesized in V. faba in response to certain stress or that endogenous ITCs induce stomatal closure in V. faba. In A. thaliana guard cells, AITC induces ROS accumulation mediated by SHAM-sensitive peroxidases, followed by NO accumulation. In the present
study, AITC also induces ROS accumulation mediated by SHAM-sensitive peroxidases, followed by NO accumulation in V. faba guard cells (Figs. 2 and 3). Cysteine, SA, indole-3-acetic acid and aromatic monoamines can be endogenous substrates of SHAMsensitive peroxidases,23–26) but it remains to be clarified what is a substrate of the SHAM-sensitive peroxidases responsible for AITC-induced ROS production. It is known that ITCs readily react with many of nucleophilic biomolecules such as proteins in animal cells because of their high electrophilicity.27) Therefore, AITC may induce signal transduction leading to stomatal closure by attacking nucleophilic targets that are conserved in plants. GSH can promptly react with ITCs to form conjugates.19) GSHmee is membrane-permeable and is converted to free GSH by cytosolic esterases.28) Therefore, application of GSHmee can diminish the intracellular AITC effects through the formation of conjugates. Treatments with GSHmee induced reopening of AITC-treated stomata and suppressed depletion of
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Fig. 2. Involvement of peroxidase-mediated ROS production in AITC-induced stomatal closure. Notes: (A) Effects of SHAM, sodium azide (NaN3), CAT, and diphenylene iodonium chloride (DPI) (gray bars) on AITC-induced stomatal closure (black bar). White bar, control; dotted bar, DMSO. Stomatal aperture widths of epidermal tissue of V. faba leaves pretreated with 2 mM SHAM, 0.2 mM NaN3, 100 units ml−1 of CAT, and 20 μM DPI for 30 min were incubated in the medium containing 50 μM AITC for 2 h. Thin solid lines with asterisks (*) indicate Student’s t-test pairs which showed significance (p < 0.05). Data represent mean ± standard errors (3 independent experiments, 60 total stomata). (B) Effects of 2 mM SHAM, 0.2 mM NaN3, 100 units ml−1 of CAT, and 20 μM DPI on stomatal aperture in V. faba (gray bars). White bar, control; divot bar, DMSO. Data are the means of three independent experiments (60 total stomata per bar). (C) AITC-induced ROS production in V. faba guard cells. The vertical scale represents the percentage of DCF fluorescent level when fluorescence intensity of the control treatment is taken as 100%. White bar, control; dotted bar DMSO; black bar, 50 μM AITC; gray bars, 50 μM AITC and 2 mM SHAM, 0.2 mM NaN3 or 100 units ml−1 of CAT. Data were obtained from 3 independent experiments (at least 60 total guard cells). Error bars represent standard errors. Asterisk (*) denotes a significant difference from solvent control (p < 0.05).
Fig. 3. Involvement of nitric oxide (NO) production in AITC-induced stomatal closure. Notes: (A) Effect of 100 μM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (gray bar) on 50 μM AITC-induced stomatal closure in V. faba (black bar). White bar, control; dotted bar, DMSO. Thin solid lines with asterisks (*) indicate pairs of Student’s t-test which exhibit significance (p < 0.05). Data represent means of three independent experiments (60 total stomata per bar). (B) Effect of 2 mM SHAM (gray bar) on 50 μM AITC-induced NO production in guard cells (black bar). The vertical scale represents the percentage of the fluorescent levels when the fluorescent intensities of treated cells are normalized to the control value taken as 100% (white bar). Dotted bar, DMSO. Each datum was obtained from at least 60 guard cells (n = 3). Error bars represent standard errors. Asterisk (*) denotes a significant difference from solvent control (p < 0.05).
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Fig. 4. Involvement of cytosolic alkalization in AITC-induced stomatal closure. Notes: (A) Effect of sodium butyrate on AITC-induced stomatal closure. White bar, control; dotted bar, DMSO; black bar, 50 μM AITC; gray bar, 50 μM AITC + 0.5 mM sodium butyrate. Thin solid lines with asterisks (*) indicate pairs of Student’s t-test which exhibit significance (p < 0.05). Data are the mean of three independent experiments (60 total stomata per bar). (B) Effect of SHAM on AITC-induced cytosolic alkalization in guard cells. The vertical scale represents the percentage of BCECF fluorescent levels normalized to control value as 100%. White bar, control; dotted bar, DMSO; black bar, 50 μM AITC; gray, 50 μM AITC + 2 mM SHAM. Each datum was obtained from at least 60 guard cells (n = 3). Error bars represent standard errors. Asterisk (*) denotes a significant difference from the solvent control (p < 0.05).
GSH in guard cells. These results suggest that the targets of AITC are present in guard cells. In the present study, AITC induces cytosolic alkalization following ROS accumulation, leading to stomatal closure in V. faba (Fig. 4). AITC also induced cytosolic alkalization in A. thaliana guard cells (data not shown). It has been reported that H2O2 induces cytosolic alkalization to close stomata.29) On the other hand, cytosolic alkalization precedes ROS accumulation in abscisic acid- and methyl jasmonate-induced stomatal closure, or occurs without ROS accumulation in methyl jasmonate-treated guard cells.5,16) Therefore, the role of cytosolic alkalization in guard cell signaling remains to be clarified. Many species of plants can launch a series of defense responses including stomatal closure in response to volatile compounds such as methyl jasmonate and ethylene.6,30) Stomatal closure in response to various stimuli is beneficial for plants since it plays important roles in stress responses such as suppressing water loss induced by wounding and inhibiting microbe infection.31,32) In this study, gaseous AITC-induced stomatal closure in V. faba (Fig. 1(B)), suggesting that AITC can serve as a volatile signal mediator for plantto-plant interaction. However, the biological significance of the interaction mediated by AITC remains unknown. It is concluded that AITC-induced ROS production mediated by SHAM-sensitive peroxidase, NO production, cytosolic alkalization, and GSH depletion in guard cells, resulting in stomatal closure in V. faba (Fig. 5). In addition, our results suggest that AITC signal pathway is not unique to AITC-producing plants such as A. thaliana but is common to plants.
Fig. 5. A simple model of AITC signaling in guard cells. AITC induces ROS accumulation, followed by NO accumulation and cytosolic alkalization, resulting in stomatal closure, which is inhibited by GSH and SHAM.
experiments and contributed to discussions. M. A. S., W. Y., and Y. M. wrote the manuscript.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was supported in part by Grants for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan [N/A].
Author contributions References M.A.S. performed the experiments. N. A., E. O., M. U., W. Y., I. C. M., and Y. N. assisted with the
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Allyl isothiocyanate induces stomatal closure in Vicia faba Muhammad Abdus Sobahan, Nasima Akter, Eiji Okuma, Misugi Uraji, Wenxiu Ye, Izumi C. Mori, Yoshimasa Nakamura & Yoshiyuki Murata To cite this article: Muhammad Abdus Sobahan, Nasima Akter, Eiji Okuma, Misugi Uraji, Wenxiu Ye, Izumi C. Mori, Yoshimasa Nakamura & Yoshiyuki Murata (2015) Allyl isothiocyanate induces stomatal closure in Vicia faba, Bioscience, Biotechnology, and Biochemistry, 79:10, 1737-1742, DOI: 10.1080/09168451.2015.1045827 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1045827
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Date: 08 November 2015, At: 19:37
Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 10, 1737–1742
Allyl isothiocyanate induces stomatal closure in Vicia faba Muhammad Abdus Sobahan, Nasima Akter, Eiji Okuma, Misugi Uraji, Wenxiu Ye, Izumi C. Mori, Yoshimasa Nakamura and Yoshiyuki Murata* Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan Received February 5, 2015; accepted April 22, 2015
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http://dx.doi.org/10.1080/09168451.2015.1045827
Isothiocyanates are enzymatically produced from glucosinolates in plants, and allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis thaliana. In this study, we investigated stomatal responses to AITC in Vicia faba. AITC-induced stomatal closure accompanied by reactive oxygen species (ROS) and NO production, cytosolic alkalization and glutathione (GSH) depletion in V. faba. GSH monoethyl ester induced stomatal reopening and suppressed AITC-induced GSH depletion in guard cells. Exogenous catalase and a peroxidase inhibitor, salicylhydroxamic acid, inhibited AITC-induced stomatal closure, unlike an NAD(P)H oxidase inhibitor, diphenylene iodonium chloride. The peroxidase inhibitor also abolished the AITC-induced ROS production, NO production, and cytosolic alkalization. AITC-induced stomatal closure was suppressed by an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide, and an agent to acidify cytosol, butyrate. These results indicate that AITC-induced stomatal closure in V. faba as well as in A. thaliana and suggest that AITC signaling in guard cells is conserved in both plants. Key words:
guard cells; peroxidase; stomatal closure
signaling;
Isothiocyanates (ITCs) that have biocidal and repellent activities against a wide range of pests are volatile organic compounds formed by the hydrolysis catalyzed by myrosinases of glucosinolates mostly in Brassicaceae plants.1) We have demonstrated that AITC, one of major ITCs, induces stomatal closure in Arabidopsis thaliana.2) It is unknown whether AITC induces stomatal responses in other plant taxa. Reactive oxygen species (ROS) and NO function as important secondary messengers in stomatal closure induced by various stimuli in a variety of plants including A. thaliana.3–8) In A. thaliana guard cells, AITC-induced ROS accumulation is mediated by salicylhydroxamic acid (SHAM)-sensitive peroxidases and AITC-induced NO production is dependent on the ROS accumulation.9) Glutathione (GSH) is the most
abundant non-protein thiol compound in plants and functions as a negative regulator in abscisic acid- and methyl jasmonate-induced stomatal closure, which are accompanied by GSH depletion in A. thaliana guard cells.10–12) In A. thaliana guard cells, AITC also induced GSH depletion and increasing intracellular GSH suppressed AITC-induced stomatal closure.2) Moreover, cytosolic alkalization is an important signaling event in stomatal closure.13–16) However, signaling mechanisms involved in AITC-induced stomatal closure in other plant taxa than A. thaliana remain to be clarified. Vicia faba belongs to Fabaceae but not to sixteen families of dicotyledonous angiosperms in which glucosinolates were present17) and is well studied as a material for stomatal movement. Hence, we employed V. faba as a material to examine stomatal responses to AITC of plants that do not belong to Brassicales order.
Materials and methods Plant materials and growth conditions. Broad bean (Vicia faba L., cv. House Ryousai) plants were grown in plastic pots (15 × 18 × 14 cm) filled with 70% (v/v) vermiculite (Asahi-Kogyo, Okayama, Japan) and 30% (v/v) Kureha soil (Kureha Chemical, Tokyo, Japan) in a growth chamber (22 ± 2 °C, 80 μmol m−2 s−1 under a 16-h-light/8-h-dark regime). Measurement of stomatal aperture. Stomatal aperture measurements were performed as described previously.18) Measurement of GSH in guard cells. Contents of GSH in guard cells were examined using monochlorobimane as previously described.10) Measurement of ROS and NO production in guard cells. Production of ROS and NO in guard cells was evaluated using 2′,7′-dichlorodihydrofluorescein
*Corresponding author. Email: [email protected]; Current address of Sobahan MA: School of Agriculture and Rural Development, Bangladesh Open University, Gazipur, Bangladesh. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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diacetate (H2DCF-DA) (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 4,5-diaminofluorescein-2 diacetate (DAF-2DA), respectively.16)
(Fig. 1(D)). These results suggest that GSH negatively regulates AITC-induced stomatal closure in V. faba as well as in A. thaliana.2)
Measurement of pHcyt. Cytosolic pH (pHcyt) in guard cells was analyzed using 2′,7′-bis(carboxyethyl)5,6-carboxyfluorescein acetomethylester (BCECF-AM) as previously described.15)
ROS are involved in AITC-induced stomatal closure in V. faba In A. thaliana guard cells, SHAM-sensitive peroxidases mediate ROS production induced by stimuli such as AITC, salicylic acid and elicitors,2,9,18) while NAD (P)H oxidases mediate ROS production induced by stimuli such as abscisic acid and methyl jasmonate.6,21) To examine the involvement of ROS in AITC-induced stomatal closure in V. faba, we examined the effects of peroxidase inhibitors, SHAM and sodium azide (NaN3), the hydrogen peroxide scavenger, catalase (CAT), and the NAD(P)H oxidase inhibitor, diphenylene iodonium chloride (DPI). As shown in Fig. 2(A), SHAM (p < 10−4), NaN3 (p < 0.001), and CAT (p < 10−4) strongly inhibited AITC-induced stomatal closure. However, the inhibition by DPI was not observed (p = 0.63). Neither SHAM (p = 0.88), NaN3 (p = 0.23), CAT (p = 0.17), nor DPI (p = 0.40) affected stomatal aperture in V. faba untreated with AITC (Fig. 2(B)). As shown in Fig. 2(C), 50 μM AITC-induced ROS accumulation in guard cells (p < 0.002). AITC failed to increase ROS level in guard cells in the presence of 2 mM SHAM (p = 0.17), NaN3 (p = 0.10), and CAT (p = 0.89) (Fig. 2(C)). These results indicate that AITC-induced stomatal closure is accompanied by ROS production mediated by SHAM-sensitive peroxidases in V. faba.
Treatment with gaseous AITC. To examine the effects of gaseous AITC, the excised leaves (15 × 10 mm) were floated on the incubation medium in an air-tight transparent plastic jar (420 ml) for 2 h in the light (80 μmol m−2 s−1) to open stomata. The desired amount of AITC (density: 1.013 g ml−1) was dropped on another dish in the jar and was spontaneously vaporized in a few minutes, and then, the leaves were incubated for another 2 h. Note that all amount of AITC was vaporized within 5 min on the dish in the jar. After the incubation, stomatal apertures were measured. Statistical analysis. Mean values were compared using Student’s t-test. Differences were considered significant for p values < 0.05.
Results AITC-induced stomatal closure accompanied by GSH depletion in V. faba Application of 10, 50, and 100 μM AITC narrowed stomatal apertures by 22% (p < 0.01), 37% (p < 10−4), and 43% (p < 10−4), respectively (Fig. 1(A)). There is no significant difference in stomata between no treatment control and the solvent control (p = 0.23). Gaseous AITC also induced stomatal closure in a dosedependent manner (Fig. 1(B)), suggesting that V. faba perceives gaseous AITC as well as dissolved AITC even though it does not produce ITCs. High concentrations of ITCs are toxic to cells. We tested viability of guard cells using fluorescein diacetate and neutral red. Guard cells treated with 10, 50, and 100 μM AITC for 2 h were stained by both dyes (data not shown), suggesting that AITC induces stomatal closure without reducing viability in V. faba. GSH can enzymatically and non-enzymatically react with ITCs,19) and membrane-permeable GSH monoethyl ester (GSHmee) can increase GSH contents in A. thaliana guard cells.10,20) To investigate whether the AITC-induced stomatal closure is reversible and whether GSH is involved in the stomatal closure in V. faba, the following experiments were performed. The epidermal tissues were incubated in the incubation medium containing AITC for 30 min to induce stomatal closure, followed by the addition of GSHmee to the medium. Subsequently, stomatal aperture width was measured (Fig. 1(C)). GSHmee significantly induced stomatal reopening (Fig. 1(C)). These results suggest that stomata of V. faba can reversibly respond to AITC. AITC decreased GSH in guard cells, which was significantly suppressed by treatment with GSHmee
NO was involved in AITC-induced stomatal closure in V. faba AITC-induced stomatal closure was suppressed by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (p < 0.001), where cPTIO alone did not affect stomatal aperture (p = 0.81) (Fig. 3(A)). As shown in Fig. 3(B), 50 μM AITC-induced NO production (p < 0.007, black bar) in guard cells, which was cancelled by 2 mM SHAM (p = 0.26, gray bar). These results indicate that NO production occurs in the process of AITC-induced stomatal closure, and the NO production depends on the SHAM-sensitive peroxidases in V. faba. Cytosolic alkalization was involved in AITC-induced stomatal closure in V. faba The AITC-induced stomatal closure was suppressed by the cytosol-acidifying agent, sodium butyrate (p < 0.001), where sodium butyrate alone did not affect stomatal aperture (p = 0.47) (Fig. 4(A)). As shown in Fig. 4(B), 50 μM AITC increased fluorescent intensity of BCECF (p < 0.004), indicating that AITC induces cytosolic alkalization in guard cells. AITC did not elicit cytosolic alkalization in guard cells when treated with 2 mM SHAM (p = 0.53) (Fig. 4(B)), suggesting that AITC-induced cytosolic alkalization depends on ROS production mediated by SHAMsensitive peroxidases.
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Fig. 1. Stomatal response to AITC in Vicia faba. Notes: (A) AITC-induced stomatal closure in V. faba. The epidermal tissues from V. faba leaves were treated with 10, 50, and 100 μM AITC for 2 h (black bars). White bar, control; dotted bar, dimethyl sulfoxide (DMSO). Data are the means of three independent experiments (60 total stomata per bar). (B) Gaseous AITC induced stomatal closure. The excised leaves of V. faba were floated on the incubation medium in an air-tight transparent plastic jar (420 ml) with 1, 5, and 10 μmol liquid AITC (black bars). Note that all amount of AITC was vaporized within 5 min on the dish in the jar. White bar, non-treatment control. Data are the means of three independent experiments (60 total stomata per bar). (C) Reopen time course of stomata pretreated with AITC by GSHmee. The epidermal preparation of V. faba leaves pretreated with 50 μM AITC for 30 min was rinsed with distilled water to remove excess AITC and incubated in the incubation medium supplemented with 10 μM GSHmee (open circles), DMSO (open triangles), or control (closed triangles). Data are the means of three independent experiments (60 total stomata per point). (D) Effects of AITC and GSHmee on GSH content in V. faba guard cells. White bar, control; divot bar, DMSO; black bar, 50 μM AITC; gray bars, 50 μM AITC and 10 μM GSHmee. Abaxial epidermal leaf tissues pre-treated with 10 μM GSHmee for 30 min were treated with 50 μM AITC. Each datum was obtained from at least 60 guard cells (n = 3). Error bars represent standard errors. Asterisk (*) denotes significant difference from the solvent control (p < 0.05).
Discussion AITC induces stomatal closure in the crucifer plant, A. thaliana, which can produce ITCs.2,9) In V. faba, AITC also induced stomatal closure (Fig. 1). This result suggests that V. faba possess a potential signal pathway leading to stomatal closure in response to AITC and that AITC-induced stomatal closure is not unique to A. thaliana but is common to plants. To our knowledge, it is unclear whether V. faba can biosynthesize glucosinolates. Separately, isoflavone, which is prevalent in legumes, is found to exist in A. thaliana although A. thaliana does not possess the genetic machinery (annotation of genome) of isoflavone synthesis.22) Hence, we cannot exclude the possibility that glucosinolates are biosynthesized in V. faba in response to certain stress or that endogenous ITCs induce stomatal closure in V. faba. In A. thaliana guard cells, AITC induces ROS accumulation mediated by SHAM-sensitive peroxidases, followed by NO accumulation. In the present
study, AITC also induces ROS accumulation mediated by SHAM-sensitive peroxidases, followed by NO accumulation in V. faba guard cells (Figs. 2 and 3). Cysteine, SA, indole-3-acetic acid and aromatic monoamines can be endogenous substrates of SHAMsensitive peroxidases,23–26) but it remains to be clarified what is a substrate of the SHAM-sensitive peroxidases responsible for AITC-induced ROS production. It is known that ITCs readily react with many of nucleophilic biomolecules such as proteins in animal cells because of their high electrophilicity.27) Therefore, AITC may induce signal transduction leading to stomatal closure by attacking nucleophilic targets that are conserved in plants. GSH can promptly react with ITCs to form conjugates.19) GSHmee is membrane-permeable and is converted to free GSH by cytosolic esterases.28) Therefore, application of GSHmee can diminish the intracellular AITC effects through the formation of conjugates. Treatments with GSHmee induced reopening of AITC-treated stomata and suppressed depletion of
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Fig. 2. Involvement of peroxidase-mediated ROS production in AITC-induced stomatal closure. Notes: (A) Effects of SHAM, sodium azide (NaN3), CAT, and diphenylene iodonium chloride (DPI) (gray bars) on AITC-induced stomatal closure (black bar). White bar, control; dotted bar, DMSO. Stomatal aperture widths of epidermal tissue of V. faba leaves pretreated with 2 mM SHAM, 0.2 mM NaN3, 100 units ml−1 of CAT, and 20 μM DPI for 30 min were incubated in the medium containing 50 μM AITC for 2 h. Thin solid lines with asterisks (*) indicate Student’s t-test pairs which showed significance (p < 0.05). Data represent mean ± standard errors (3 independent experiments, 60 total stomata). (B) Effects of 2 mM SHAM, 0.2 mM NaN3, 100 units ml−1 of CAT, and 20 μM DPI on stomatal aperture in V. faba (gray bars). White bar, control; divot bar, DMSO. Data are the means of three independent experiments (60 total stomata per bar). (C) AITC-induced ROS production in V. faba guard cells. The vertical scale represents the percentage of DCF fluorescent level when fluorescence intensity of the control treatment is taken as 100%. White bar, control; dotted bar DMSO; black bar, 50 μM AITC; gray bars, 50 μM AITC and 2 mM SHAM, 0.2 mM NaN3 or 100 units ml−1 of CAT. Data were obtained from 3 independent experiments (at least 60 total guard cells). Error bars represent standard errors. Asterisk (*) denotes a significant difference from solvent control (p < 0.05).
Fig. 3. Involvement of nitric oxide (NO) production in AITC-induced stomatal closure. Notes: (A) Effect of 100 μM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (gray bar) on 50 μM AITC-induced stomatal closure in V. faba (black bar). White bar, control; dotted bar, DMSO. Thin solid lines with asterisks (*) indicate pairs of Student’s t-test which exhibit significance (p < 0.05). Data represent means of three independent experiments (60 total stomata per bar). (B) Effect of 2 mM SHAM (gray bar) on 50 μM AITC-induced NO production in guard cells (black bar). The vertical scale represents the percentage of the fluorescent levels when the fluorescent intensities of treated cells are normalized to the control value taken as 100% (white bar). Dotted bar, DMSO. Each datum was obtained from at least 60 guard cells (n = 3). Error bars represent standard errors. Asterisk (*) denotes a significant difference from solvent control (p < 0.05).
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Fig. 4. Involvement of cytosolic alkalization in AITC-induced stomatal closure. Notes: (A) Effect of sodium butyrate on AITC-induced stomatal closure. White bar, control; dotted bar, DMSO; black bar, 50 μM AITC; gray bar, 50 μM AITC + 0.5 mM sodium butyrate. Thin solid lines with asterisks (*) indicate pairs of Student’s t-test which exhibit significance (p < 0.05). Data are the mean of three independent experiments (60 total stomata per bar). (B) Effect of SHAM on AITC-induced cytosolic alkalization in guard cells. The vertical scale represents the percentage of BCECF fluorescent levels normalized to control value as 100%. White bar, control; dotted bar, DMSO; black bar, 50 μM AITC; gray, 50 μM AITC + 2 mM SHAM. Each datum was obtained from at least 60 guard cells (n = 3). Error bars represent standard errors. Asterisk (*) denotes a significant difference from the solvent control (p < 0.05).
GSH in guard cells. These results suggest that the targets of AITC are present in guard cells. In the present study, AITC induces cytosolic alkalization following ROS accumulation, leading to stomatal closure in V. faba (Fig. 4). AITC also induced cytosolic alkalization in A. thaliana guard cells (data not shown). It has been reported that H2O2 induces cytosolic alkalization to close stomata.29) On the other hand, cytosolic alkalization precedes ROS accumulation in abscisic acid- and methyl jasmonate-induced stomatal closure, or occurs without ROS accumulation in methyl jasmonate-treated guard cells.5,16) Therefore, the role of cytosolic alkalization in guard cell signaling remains to be clarified. Many species of plants can launch a series of defense responses including stomatal closure in response to volatile compounds such as methyl jasmonate and ethylene.6,30) Stomatal closure in response to various stimuli is beneficial for plants since it plays important roles in stress responses such as suppressing water loss induced by wounding and inhibiting microbe infection.31,32) In this study, gaseous AITC-induced stomatal closure in V. faba (Fig. 1(B)), suggesting that AITC can serve as a volatile signal mediator for plantto-plant interaction. However, the biological significance of the interaction mediated by AITC remains unknown. It is concluded that AITC-induced ROS production mediated by SHAM-sensitive peroxidase, NO production, cytosolic alkalization, and GSH depletion in guard cells, resulting in stomatal closure in V. faba (Fig. 5). In addition, our results suggest that AITC signal pathway is not unique to AITC-producing plants such as A. thaliana but is common to plants.
Fig. 5. A simple model of AITC signaling in guard cells. AITC induces ROS accumulation, followed by NO accumulation and cytosolic alkalization, resulting in stomatal closure, which is inhibited by GSH and SHAM.
experiments and contributed to discussions. M. A. S., W. Y., and Y. M. wrote the manuscript.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was supported in part by Grants for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan [N/A].
Author contributions References M.A.S. performed the experiments. N. A., E. O., M. U., W. Y., I. C. M., and Y. N. assisted with the
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