Hydrogen peroxide is involved in methyl jasmonate-induced
senescence of rice leaves
Kuo Tung Hung, Yi Ting Hsu and Ching Huei Kao*
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China
Correspondence *Corresponding author, e-mail: [email protected]
Received 6 August 2005; revised 25 November 2005
doi: 10.1111/j.1399-3054.2006.00662.x
The role of H2O2 in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H2O2 production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H2O2, was observed to be effective in inhibiting MJ-induced senescence and MJ-increased mal-ondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ-induced H2O2 production, suggesting that NADPH oxidase is a H2O2 -generating enzyme in MJ-treated detached rice leaves. DPI and IMD also inhibited MJ-promoted senescence and MJ-increased MDA content in detached rice leaves. Phosphatidylinositol 3-kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ-induced H2O2production and senes-cence of detached rice leaves. Exogenous H2O2reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)-sensitive and those of cultivar Tainung 67 (TNG67) are JA-insensitive. On treatment with JA, H2O2accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ-induced H2O2 produc-tion in detached rice leaves is abscisic acid (ABA)-independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ- and ABA-induced H2O2production and senescence of detached rice leaves, suggest-ing that the action of MJ and ABA is ethylene-dependent.
Introduction
Methyl jasmonate (MJ) was first considered as one of many plant secondary metabolites with a possible appli-cation in the perfume industry (Demole et al. 1962). It is now becoming evident that jasmonates can act as true plant hormones, which mediate in various aspects of development and stress responses (Creelman and Mullet 1997). It has been shown that jasmonates are powerful
promoters of leaf senescence (Ueda and Kato 1981, Weidhase et al. 1987, Chou and Kao 1992, Beltrano et al. 1998, Hung and Kao 1998, 2004a). Recent mole-cular studies have confirmed that jasmonic acid (JA) may play a role in leaf senescence (He et al. 2002).
Active oxygen species (AOS)-initiated lipid peroxida-tion has been thought to be an important mechanism of
Abbreviations – ABA, abscisic acid; AOS, active oxygen species; DAB, 3,3-diaminobenzidine; DMTU, dimethylthiourea; DPI, diphenyleneiodonium chloride; ELISA, enzyme-linked immunosorbent assay; FW, fresh weight; IMD, imidazole; LY, LY 294002; MDA, malondialdehyde; MJ, methyl jasmonate; PI3K, phosphatidylinositol kinase; PI3P, phosphatidylinositol 3-phosphate; TN1, Taichung Native 1; TNG67, Tainung 67; WM, wortmannin.
leaf senescence (Kellogg and Fridovich 1975, Thompson et al. 1987). It has been shown that MJ causes the generation of H2O2 (Orozco-Ca´rdenas and Ryan 1999, Orozco-Ca´rdenas et al. 2001, Hung and Kao 2004a) and lipid peroxidation expressed as malon-dialdehyde (MDA) production in plant cells (Hung and Kao 1998, 2004a). Thus, MJ leads to oxidative stress in plant cells.
H2O2is a constituent of oxidative metabolism and is itself an AOS. It has been shown that H2O2 promotes leaf senescence (Begam and Choudhuri 1992, Lin and Kao 1998) and induction of senescence is accompanied by an increase in endogenous H2O2 content (Mondal and Choudhuri 1981, Hung and Kao 2003, 2004a). Because H2O2is relatively stable and diffusible through membrane, H2O2 is thought to constitute a general signal molecule inducing cellular stress (Foyer et al. 1997, Neill et al. 2002). Thermoprotection obtained by spraying salicylic acid or by heat acclimation was sug-gested to be achieved by a common signal transduction pathway involving very early increase in H2O2content (Dat et al. 1998). In tomato plants, H2O2 has been shown to act as a second messenger for the induction of defense genes in response to wounding, systemin, and MJ (Orozco-Ca´rdenas et al. 2001). MJ has also been shown to generate H2O2 in parsley suspension-cultured cells (Kauss et al. 1994). It has been demon-strated that H2O2 is required for the induction of rice cytosolic ascorbate peroxidase mRNA (Morita et al. 1999). H2O2has now also been shown to be a crucial component of abscisic acid (ABA)-induced stomatal clo-sure (Pei et al. 2000, Zhang et al. 2001, Kwak et al. 2003) and gibberellic acid-induced programmed cell death in the aleurone cells of barley (Fath et al. 2001).
We have previously shown that MJ not only increases the contents of H2O2but also causes protein loss (senes-cence) and lipid peroxidation in rice leaves (Hung and Kao 2004a). These results suggest that MJ causes oxida-tive stress and MJ-promoted senescence is mediated through oxidative stress. In this article, we have exam-ined the role of H2O2as a link between MJ and subse-quent senescence in detached rice leaves.
Materials and methods Plant materials
Rice (Oryza sativa L., cv. Taichung Native 1, TN1, or Tainung 67, TNG67) seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed extensively with distilled water. These seeds were then germinated in Petri dishes with wetted filter paper at 37C under dark conditions. After 48-h incubation, uniformly
germinated seeds were selected and cultivated in a 500-ml beaker containing half-strength Kimura B solu-tion as described previously (Hsu and Kao 2005). The hydroponically cultivated seedlings were grown for 12 days in a Phytotron (Agricultural Experimental Station, National Taiwan University, Taipei, Taiwan) with nat-ural sunlight at 30C day/25C night and 90% relative humidity. The apical 3 cm of the third leaf of TN1 was used in experiments. A group of 10 segments was floated in a Petri dish containing 10 ml of test solution. Incubation was carried out at 27C in the dark. For experiments of intact leaves of TN1 and TNG67 seed-lings, JA was added to half-strength Kimura B solution at the time when the third leaf was fully expanded. Determinations of protein, H2O2, lipid peroxidation, and ABA
The senescence of detached rice leaves was followed by measuring the decrease of protein content. For protein extraction, leaf segments were homogenized in 50 mM sodium phosphate buffer (pH 6.8). The extracts were centrifuged at 17 600 g for 20 min, and the supernat-ants were used for determination of protein by the method of Bradford (1976).
The H2O2content was measured colorimetrically as described by Jana and Choudhuri (1982). H2O2 was extracted by homogenizing leaf tissue with phosphate buffer (50 mM, pH 6.5) containing 1 mM hydroxyla-mine. The homogenate was centrifuged at 6000 g for 25 min. To determine H2O2 content, the extracted solution was mixed with 0.1% titanium chloride in 20% (v/v) H2SO4. The mixture was then centrifuged at 6000 g for 25 min. The absorbance was measured at 410 nm. Using this method, we obtained that absorb-ance increased linearly with the amount of H2O2and addition of H2O2to extracts resulted in the predicted increase of absorbance, i.e. added H2O2 was fully recovered (data not shown). The H2O2 content in leaf extracts was calculated using the extinction coef-ficient of 0.28 mmol1cm1. In some experiments, H2O2 was also visually detected in the leaves by using 3,3-diaminobenzidine (DAB) as substrate (Orozco-Ca´rdenas and Ryan 1999). Detached rice leaves were supplied through the cut ends with DAB (1 mg ml1) solution for 24 h under light at 27C. Leaves were then decolorized in boiling ethanol (95%) for 0.5 h. This treatment decolorized the leaves except for the brown polymerization product pro-duced by DAB with H2O2. After cooling, the leaves were extracted at room temperature with fresh etha-nol. The H2O2staining was repeated four times with similar results.
MDA, routinely used as an indicator of lipid peroxida-tion, was extracted with 5% (w/v) trichloroacetic acid and determined according to Heath and Packer (1968).
For extraction of ABA, leaves were homogenized with a pestle and mortar in extraction solution (80% metha-nol containing 2% glacial acetic acid). To remove plant pigments and other non-polar compounds which could interfere in the immunoassay, extracts were first passed through polyvinylpyrrolidone column and C18 (Sep-PakÒ.Vac) cartridges (Waters, Milford, MA). The eluates were concentrated to dryness by vacuum evaporation and resuspended in Tris-buffered saline before enzyme-linked immunosorbent assay (ELISA). ABA was quantified by ELISA (Walker-Simmons 1987). ABA immunoassay detection kit (PGR-1) purchased from Sigma Chemical Co. (St Louis, MO) is specific to (þ)–ABA. By evaluating 3H-ABA recovery, ABA loss was <3% by the method described here.
Preparation of silver thiosulfate
A stock solution of silver thiosulfate (STS) was prepared by mixing equal volume of 0.01 M AgNO3and 0.04 M Na2S2O3(Liu et al. 1990).
Statistical analysis
Statistical differences between measurements (n 5 4) on different treatments or on different times were ana-lyzed following the Duncan’s multiple range test or Student’s t-test.
Results
MJ increases H2O2production and promotes senescence
Senescence of detached rice leaves in the present study was followed by measuring the decrease of protein. MDA is used as an indicator of lipid peroxidation. The changes in protein and MDA contents in detached rice leaves treated with 45 mM MJ in the dark are shown in Fig. 1A, B. The decrease in protein content and increase in MDA content were evident 24 h after MJ treatment. Clearly, MJ is effective in promoting senescence and causing lipid peroxidation. Lipid peroxidation is caused by AOS (Kellogg and Fridovich 1975, Thompson et al. 1987). MJ also caused an increase in H2O2 content (Figs 1C and 2). The increase in H2O2 was evident 12 h after treatment of MJ, which was prior to the decrease in protein and increase in MDA. These results suggest that H2O2may play an important role in regu-lating the senescence of rice leaves induced by MJ.
The effect of dimethylthiourea, a chemical trap for H2O2
To demonstrate the involvement of H2O2 in the effects induced by MJ in detached rice leaves, namely the decrease in protein content and the increase in MDA content, dimethylthiourea (DMTU), a chemical trap for H2O2 (de Agazio and Zacchini 2001), was used. Detached rice leaves were incubated in a solution
Protein (mg g –1 FW) 0 20 40 60 MDA (nmol g –1 FW) 0 10 20 30 40 50 Time (h) 0 12 24 36 48 H2 O2 ( µ mol g –1 FW) 0 10 20 30 40 50 MJ H2O * * * * * * * * * * A B C
Fig. 1. Changes in the contents of protein (A), malondialdehyde (MDA) (B), and H2O2(C) in detached rice leaves treated with methyl jasmonate
(MJ). Detached rice leaves were treated with either water or 45 mM MJ in the dark. Values are mean SE (n 5 4). The missing error bars indicate that they are smaller than the label marks. Asterisk represents values that are significantly different between H2O and MJ treatments
containing MJ (45 mM) with or without DMTU (5 mM). The decrease in protein and the increase in MDA in rice leaves caused by MJ were reduced by DMTU (Fig. 3A, B).
The effect of NADPH oxidase inhibitors
AOS, originating from the plasma membrane NADPH oxidase, which transfers electrons from cytoplasmic NADPH to O2 to form O2–, followed by dismutation of O2– to H2O2, has been a recent focus in AOS signal-ing. There are reports, indicating that the oxidative burst and the accumulation of H2O2appear to be mediated by the activation of plasma membrane NADPH oxidase complex in plants (Ogawa et al. 1997, del Rı´o et al.
1998, Potikha et al. 1999, Jiang and Zhang 2002). Some chemical inhibitors of the NADPH oxidase complex found in mammalian neutrophils, such as diphenyle-neiodonium chloride (DPI) and imidazole (IMD), inhibit the pathogen-, elicitor-, wound-, and abscisic acid-induced accumulation of H2O2in plants (Levine et al. 1994, Auh and Murphy 1995, Bestwick et al. 1997, Alvarez et al. 1998, Orzco-Ca´rdenas and Ryan 1999, Jiang and Zhang 2002). When detached rice leaves were treated with a solution of DPI (1 mM) or IMD (100 mM), MJ-induced accumulation of H2O2 in rice leaves was reduced (Figs 2 and 4C). DPI or IMD also inhibited MJ-promoted leaf senescence (Fig. 4A) and MJ-increased contents of MDA (Fig. 4B).
The effect of phosphatidylinositol 3-kinase inhibitors
Recently, Jung et al. (2002) and Park et al. (2003) suggested that phosphatidylinositol 3-phosphate
Time (h) 0 12 24 36 48 H2O Time (h) 12 24 36 48 MJ Time (h) 48 48 MJ+DPI MJ+IMD
Fig. 2. Histochemical detection of H2O2with DAB staining in detached
rice leaves treated with water, methyl jasmonate (MJ), and MJ plus diphenyleneiodonium chloride (DPI) or imidazole (IMD). The concentra-tions of MJ, DPI, and IMD were 45, 1, and 100 mM, respectively. DAB, 3,3-diaminobenzidine. Protein (mg –1 g FW) 0 10 20 30 40 50 MDA (nmol g –1 FW) 0 10 20 30 40 50 H2O DMTU MJ +DMTU MJ A B d c a b d a b c
Fig. 3. Effect of dimethylthiourea (DMTU) on the contents of protein (A) and malondialdehyde (MDA) (B) in detached rice leaves treated with methyl jasmonate (MJ). The concentrations of MJ and DMTU were 45 mM and 5 mM, respectively. All measurements were determined 2 days after treatment in the dark. Values are mean SE(n 5 4). Values with the same letter are not significantly different at P < 0.05 level, according to Duncan’s multiple range test.
(PI3P) is important in NADPH oxidase-mediated H2O2 production during ABA-induced stomatal closure. Thus, it is of great interest to understand whether PI3P is also important in MJ-induced H2O2production and senescence of rice leaves. LY 294002 (LY) and wortmannin (WM) are inhibitors of phosphatidylinosi-tol 3-kinase (PI3K), a product of which is PI3P. When detached rice leaves were treated with a solution of
LY (100 mM) or WM (1 mM), MJ-induced accumula-tion of H2O2 in detached rice leaves was reduced (Fig. 5C). LY or WM also inhibited MJ-promoted senescence (Fig. 5A) and MJ-increased MDA content (Fig. 5B). Exogenous H2O2(1 mM) was observed to be able to reverse the inhibitory effects of LY or WM on MJ-induced senescence and lipid peroxidation (Fig. 5A, B). Protein (mg g –1 FW) 0 10 20 30 40 50 A B C MDA (nmol g –1 FW) 0 20 40 60 H2 O2 ( µ mol g –1 FW) 0 10 20 30 40 50 c c c a b b b a a a c d a a a b b c H2O DPI IMD MJ MJ +DPI MJ +IMD
Fig. 4. Effect of diphenyleneiodonium chloride (DPI) and imidazole (IMD) on the contents of protein (A), malondialdehyde (MDA) (B), and H2O2(C) in detached rice leaves treated with methyl jasmonate (MJ).
The concentrations of MJ, DPI, and IMD were 45, 1, and 100 mM, respectively. All measurements were determined 2 days after treatment in the dark. Values are mean SE(n 5 4). Values with the same letter
are not significantly different at P < 0.05 level, according to Duncan’s multiple range test.
Protein (mg g –1 FW) 0 10 20 30 40 50 H2 O2 ( µ mol g –1 FW) 0 10 20 30 40 MDA (nmol g –1 FW) 0 10 20 30 40 50 c b b a a a b b d c c a a a a a a b c d d d A B C c c MJ – – – + + + + + LY – + – – + – + – WM – – + – – + – + H2O2 – – – – – – + +
Fig. 5. Effect of LY 294002 (LY) and wortmannin (WM) on the con-tents of protein (A), malondialdehyde (MDA) (B), and H2O2(C) in methyl
jasmonate (MJ)-treated rice leaves in the presence or absence of hydro-gen peroxide. The concentrations of LY, WM, MJ, and hydrohydro-gen per-oxide were 100 mM, 1 mM, 45 mM, and 1 mM, respectively. All measurements were determined 2 days after treatment in the dark. Values are mean SE(n 5 4). Values with the same letter are not significantly different at P < 0.05 level, according to Duncan’s multiple range test.
JA induces H2O2accumulation in the leaves of cultivar TN1 seedlings but not in cultivar TNG67 Figure 6 shows the effect of JA concentrations, in the range from 5 to 40 mM, applied over a period of 3 days, on the senescence of the second leaves of rice seed-lings. It is clear that increasing concentration of JA progressively promotes senescence of the second leaves of TN1, but not in TNG67 seedlings. It appears that in terms of leaf senescence, TNG67 is JA-insensitive and TN1 is JA-sensitive. If H2O2 is important in regulating rice leaf senescence, then H2O2content is expected to increase in JA-treated TN1 seedlings but not in TNG67. As indicated in Fig. 7 (A, C), it is indeed the case. It is also clear from Fig. 7 (B, D) that JA increased MDA content in the second leaves of TN1 but not in TNG67. ABA but not MJ increases endogenous ABA content in detached rice leaves
In our recent work, we reported that H2O2is necessary for ABA-induced senescence of detached rice leaves (Hung and Kao 2004b). To determine whether MJ-induced H2O2 production and MJ-promoted senescence of detached rice leaves are mediated through an accumulation of ABA, we measured endogenous ABA content in ABA- and MJ-treated detached rice leaves. It is clear that ABA but not MJ treatment caused an increase in the content of endogen-ous ABA in detached rice leaves (Fig. 8). It appears that
MJ-induced H2O2 production and MJ-promoted senes-cence in detached rice leaves are ABA-independent. An increase in ethylene sensitivity is associated with MJ- and ABA-increased H2O2production in detached rice leaves
A change in ethylene sensitivity in MJ- or ABA-treated detached rice leaves was tested by using STS, an inhi-bitor of ethylene action. It was observed that treatment of detached rice leaves with STS inhibited the increase in H2O2 and MDA contents (Fig. 9A, B) and the decrease in protein content (Fig. 9C) caused by MJ and ABA, suggesting that ethylene action is required for ABA- and MJ-induced H2O2production.
Discussion
It has been shown that MJ is able to generate H2O2in parsley suspension-cultured cells (Kauss et al. 1994) and tomato leaves (Orozco-Ca´rdenas and Ryan 1999, Orozco-Ca´rdenas et al. 2001). Here, we also show that MJ induced H2O2 production in rice leaves (Figs 1C and 2). Wounding is known to induce H2O2 production (Orozco-Ca´rdenas and Ryan 1999). When detached rice leaves are used to study senescence, wounding is always a problem. However, in the present study, each long and narrow rice leaf was cut
TN1
TNG67
JA (
µM
)
0
5
10
20
40
0
5
10
20
40
Fig. 6. Effect of jasmonic acid (JA) on the senescence of the second leaves of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings. Rice seedlings were cultivated in half-strength Kimura B solution in a Phytotron with natural sunlight at 30C
day/25C night and 90% relative
humidity. JA was added to half-strength Kimura B solution at the time when the third leaves were fully expanded. Arrows indicate the second leaves. Pictures were taken 3 days after adding JA.
transversely; thus, the area of wounding was very small. Therefore, H2O2 generation of detached rice leaves induced by MJ is unlikely to be complicated by the wounding effect.
A role for plasma membrane NADPH oxidase in the generation of the H2O2 has been a recent focus in AOS signaling. Here, we show that DPI and IMD, inhibitors of NADPH oxidase (Levine et al. 1994, Auh and Murphy 1995, Bestwick et al. 1997, Alvarez et al. 1998, Orozco-Ca´rdenas et al. 2001, Jiang and Zhang 2002, Kwak et al. 2003), reduced MJ-induced H2O2 production (Fig. 5C) and lipid peroxi-dation (Fig. 5B), and MJ-promoted senescence (Fig. 5A) in rice leaves. Similar results were obtained by using DMTU, a chemical trap for H2O2 (Fig. 3). Furthermore, the increase in H2O2content by MJ was observed to precede the occurrence of leaf senescence and lipid peroxidation (Fig. 1). It appears that H2O2is involved in MJ-induced senescence of detached rice leaves. In the present study, JA is effective in inducing senescence of the leaves of TN1 rice seedlings but not in TNG67, suggesting that in terms of senescence, TN1 is JA-sensitive and TNG67 is JA-insensitive (Fig. 6). The fact that on treatment with JA, the H2O2 content increased in JA-sensitive TN1 seedling leaves but not
in JA-insensitive TNG67 (Fig. 7) supports further that H2O2 is involved in leaf senescence caused by jasmonates.
It has been shown that a high concentration of DPI can affect other enzymes potentially involved in the generation of AOS, including cell wall peroxidase and nitric oxide synthase (Bolwell et al. 1998). The fact that MJ-induced H2O2accumulation in detached rice leaves can be inhibited by low concentration (1 mM) of DPI and can be inhibited by both DPI and IMD (Figs 2 and 4C) strongly suggests that MJ-dependent H2O2 genera-tion originated, at least in part, from plasma membrane NADPH oxidase.
The mechanism of AOS production and the mole-cules involved have been well investigated in animal cells, particularly in neutrophils. The NADPH oxidase complex, which consists of many components, is responsible for AOS production in neurophil cells and is activated by the binding of PI3P to one of the compon-ents (Ellson et al. 2001). It appears that PI3P is impor-tant in MJ-induced H2O2production based on two lines of evidence. First, LY or WM, inhibitors of PI3K, was able to reduce MJ-induced H2O2 production, senes-cence, and lipid peroxidation (Fig. 5). Second, exogen-ous H2O2 reversed the inhibitory effect of the PI3K JA(µM ) 0 10 20 30 40 JA(µM ) 0 10 20 30 40 H2 O2 ( µ mol g –1 FW) MDA (nmol g –1 FW) 0 10 20 30 40 0 10 20 30 40 A B D C TN1 TNG67 a b b a c b a a a a a a a a a a b c c d
Fig. 7. Effect of jasmonic acid (JA) on the contents of H2O2(A and C)
and malondialdehyde (MDA) (B and D) in the second leaves of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seed-lings. Rice seedlings were culti-vated in half-strength Kimura B solution in a Phytotron with nat-ural sunlight at 30C day/25C
night and 90% relative humidity. JA was added to half-strength Kimura B solution at the time when the third leaves were fully expanded. H2O2and MDA
con-tents were determined 3 days after adding JA. Values are mean SE(n 5 4). Values with the same letter are not signifi-cantly different at P < 0.05 level, according to Duncan’s multiple range test.
inhibitors on the MJ-induced H2O2accumulation, senes-cence, and lipid peroxidation. These results supported further the conclusion that H2O2 is involved in MJ-induced senescence of detached rice leaves and that NADPH oxidase is involved in MJ-induced H2O2 pro-duction. PI3P has also been shown to be important in the ABA-induced H2O2generation in guard cells (Jung et al. 2002, Park et al. 2003). Our recent observation also demonstrated that PI3P is required for ABA-induced H2O2 production in detached rice leaves (Hung and Kao 2005). In neutrophils, PI3P regulated H2O2 produc-tion by binding to the non-catalytic component p40phox of the NADPH oxidase (Ellson et al. 2001). However, a rice homolog of p40phox has not been reported. Therefore, the detailed mechanism of the action of PI3P during H2O2production in the rice leaves awaits further investigation.
Recently, a cell wall peroxidase has been identified in French bean (Bolwell et al. 1998, Blee et al. 2001), and a potentially peroxidase-mediated H2O2production has been demonstrated in Arabidopsis cultures challenged with a fungal elicitor (Bolwell et al. 2002). As DPI and IMD did not completely inhibit MJ-induced H2O2 pro-duction (Figs 2 and 4C), peroxidase may be another H2O2-generating enzyme operating in MJ-treated rice leaves.
In plants, polyamines are known to play an important role in growth, development, and stress responses (Walden et al. 1997). It has been shown that H2O2 produced by diamine or polyamine oxidase induced hypersensitive cell death in plants (Yoda et al. 2003). Previously, we have shown that MJ treatment increased
putrescine, a diamine, content but had no effect on spermidine and spermine contents in rice leaves (Chen et al. 1994). It appears that diamine or polya-mine oxidase is unlikely to be affected by MJ in rice leaves. An alternative source for H2O2 generation includes oxalate oxidase, an enzyme that degrades
Time (days) 0 1 2 3 ABA (pmol g –1 FW) 0 200 400 600 800 1000 ABA MJ H2O * * *
Fig. 8. Changes in the contents of endogenous abscisic acid (ABA) in detached rice leaves treated with methyl jasmonate (MJ) and ABA. The concentrations of MJ and ABA were 45 mM. Values are mean SE
(n 5 4). The missing error bars indicate that they are smaller than the label marks. Asterisk represents values that are significantly different between H2O and ABA treatments at P < 0.05 level by Student’s t-test
when compared to water control.
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 H2O STS ABA ABA +STS MJ MJ +STS a a c b d c a a b c c d d d b c a b A B C H2 O2 (µ mol g –1 FW) MDA (nmol g –1 FW) Protein (mg g –1 FW)
Fig. 9. Effect of silver thiosulfate (STS) on the contents of H2O2(A),
malondialdehyde (MDA) (B), and protein (C) of detached rice leaves in the presence or absence of abscisic acid (ABA) and methyl jasmonate (MJ). The concentrations of STS, MJ, and ABA were 200, 45, and 45 mM, respectively. All measurements were determined 2 days after treatment in the dark. Values are mean SE(n 5 4). Values with the same letter are not significantly different at P < 0.05, according to Duncan’s multiple range test.
oxalate to CO2and H2O2(Dumas et al. 1995). Oxalate oxidase gene expression is induced by salt stress, sal-icylic acid, and MJ in barley roots (Hurkman and Tanaka 1996). It is not known whether MJ will activate oxalate oxidase in rice leaves. Further work is required to clarify this possibility.
It appears that when detached rice leaves are treated with MJ, H2O2 is produced in the apoplast. Because apoplast has only a small proportion of the cell’s antiox-idant capacity, H2O2 will rapidly move into the cell to exert its effect on senescence. It has been suggested that peroxiporins or water channels (aquaporins) may serve as conduits for trans-membrane H2O2transport (Neill et al. 2002). Thus, H2O2can function as a mobile signal in MJ-treated detached rice leaves, but whether H2O2is the sole signal remains to be determined.
Our recent work demonstrated that H2O2 is also involved in ABA-induced senescence of rice leaves (Hung and Kao 2004b). Our data revealed that MJ treat-ment did not result in an accumulation of ABA in detached rice leaves (Fig. 8), suggesting that MJ-induced H2O2production is ABA-independent.
There are reports, showing that ethylene is the major senescence-promoting hormone (Gepstein and Thimann 1982, Kao and Yang 1983, Preger and Gepstein 1985). Several lines of evidence indicated that MJ-promoted leaf senescence was independent of ethylene (Abeles et al. 1989, Cuello et al. 1990). In the present study, we observed that ABA- and MJ-induced H2O2production, lipid peroxidation, and senescence in detached rice leaves are dependent on the ethylene sensitivity (Fig. 9A–C). It appears that in detached rice leaves, one of the earliest events following MJ or ABA treatment is modulating ethylene sensitivity, which then causes the production of H2O2 and the subsequent senescence promotion (Fig. 10).
Acknowledgements – This study was supported by grant NSC 93-2313-B-002-062 from the National Science Council of the Republic of China.
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