c
1998 Kluwer Academic Publishers. Printed in the Netherlands.
Involvement of lipid peroxidation in methyl jasmonate-promoted senescence
in detached rice leaves
Kuo Tung Hung & Ching Huei Kao
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China (
author for correspondence)
Received 16 September 1997; accepted in revised form 22 September 1997
Key words: leaf senescence, lipid peroxidation, methyl jasmonate, Oryza sativa
Abstract
Lipid peroxidation in relation to MJ-promoted senescence of detached rice leaves was investigated. Lipid peroxi-dation seems to be involved in the regulation of MJ-promoted senescence of detached rice leaves. This conclusion was based on the observations that (a) methyl jasmonate, which was found to promote senescence, increases the level of lipid peroxidation, (b) linolenic and linoleic acid, precursors of the biosynthesis of jasmonic acid, promoted senescence and increased lipid peroxidation level, (c) benzyladenine, a synthetic cytokinin, counteracted MJ-promoted senescence and reduced the increase in lipid peroxidation level, (d) calcium chloride effectively reduced MJ-promoted senescence and at the same time reduced MJ-promoted lipid peroxidation. Free radical scav-engers (reduced glutathione and sodium benzoate) and an iron chelator (2, 20
-bipyridine) prevented MJ-promoted senescence, suggesting that lipid peroxidation induced by MJ is mediated through free radicals.
Abbreviations: BA = benzyladenine; BP = 2, 20
-bipyridine; GSH = reduced glutathione; MDA = malondialdehyde; MJ = methyl jasmonate; SB = sodium benzoate
1. Introduction
Jasmonates are endogenous substances that have been identified in many plants [12]. Jasmonates were found to be powerful promoter of leaf senescence [2, 8, 9, 17, 19]. Lipid peroxidation is considered to be an important mechanism of leaf senescence [3, 11, 15, 16]. Dhindsa et al. [4] demonstrated that the inhibition of senescence of detached oat and Rumex leaves by plant hormones was mediated through modulation of free radical-induced lipid peroxidation. In this study, effects of MJ, and linoleic and linolenic acids, pre-cursors of jasmonic acid biosynthesis [18], on degra-dation of chlorophyll and protein and lipid peroxida-tion were investigated. Furthermore, the effects of two free radical scavengers (glutathione, GSH, and sodium benzoate, SB) [14, 21] and one metal chelator (2,20
-bipyridine, BP) on degradation of chlorophyll and pro-tein were also examined.
2. Materials and methods
Rice (Oryza sativa cv. Taichung Native 1) was cultured as previously described [10]. The apical 3-cm seg-ments excised from the third leaves of 12-day-old seedlings were used. A group of 10 segments was floated in a Petri dish containing 10 ml of test solu-tions. Incubation was carried out at 27
C in darkness. Chlorophyll was determined according to Winter-mans and De Mots [20]. After extraction in 96% (v/v) ethanol. For protein extraction, leaf segments were homogenized in 25 mM sodium phosphate buffer (pH 7.5). The extracts were centrifuged at 17,000 g for 20 min, and the supernatants were used for determi-nation of protein by the method of Bradford [1]. Pro-tein and chlorophyll levels were expressed as mg g 1
fresh weight. MDA was extracted with 5% (w/v) trichloroacetic acid and determined according to Heath and Packer [7]. MDA level was expressed as nmol g 1
18
fresh weight and is routinely used as an index of lipid peroxidation.
For the experiments of the effect of linoleic and linolenic acids (5 mM) on chlorophyll, protein and MDA levels in detached rice leaves, all treatments included 0.1% Tween 20. Chlorophyll, protein and MDA levels were determined after 4 days in darkness. For those experiments with treatments that includ-ed GSH (30 mM) or SB (10 mM) together with MJ (45M), chlorophyll, protein and MDA levels were determined after 3 days in darkness. As to the effect of BP (1 mM) on chlorophyll and protein levels in detached rice leaves treated with MJ (45M), chloro-phyll, protein and MDA levels were determined after 3 days in darkness.
MJ [(–)–MJ] was a product of Serva and other chemicals were purchased from Sigma Chemical Co.
3. Results and discussion
The senescence of detached rice leaves in the dark was followed by measuring the decrease of chloro-phyll and protein. Changes in the levels of chlorochloro-phyll, protein, and MDA in detached rice leaves treated with 45M MJ are shown in Figure 1. It is clear that the pro-motion of chlorophyll loss and protein degradation by MJ was evident one day after MJ treatment. MDA level in MJ-treated detached rice leaves was observed to be higher than that in water-treated controls throughout the entire duration of incubation. This shows that MJ-promoted senescence of detached rice leaves is linked to lipid peroxidation.
It has been shown that cytokinins, known to delay senescence, counteract jasmonic acid effect with respect to chlorophyll degradation in detached barley leaves [19]. In detached rice leaves, we observed that BA, a synthetic cytokinin, counteracted MJ-promoted senescence (Figure 2). Figure 2 also shows that BA reduced the increase in MDA level in detached rice leaves treated with MJ.
Previously, we have shown that calcium interacts with jasmonates in the regulation of senescence of detached rice leaves [2]. In the present study, we found that treatment with calcium chloride effectively reduced MJ-promoted senescence and at the same time reduced MJ-increased lipid peroxidation (Figure 3).
It has been shown that oxidation of linolenic acid by lipoxygenase is the first step in the biosynthesis path-way of jasmonic acid [18]. Recently, we demonstrated that linoleic acid (18:2) and linolenic acid (18:3)
pro-Figure 1. Changes in chlorophyll, protein, and MDA levels in
detached rice leaves treated with MJ. The concentration of MJ was 45M. Bars indicate SE (n = 4).
moted senescence of detached rice leaves [9]. Here, we reported that linoleic and linolenic acid -promoted
Figure 2. Effect of BA on MJ-promoted senesence and MJ-increased
MDA level in detached rice leaves. Chlorophyll, protein, and MDA levels were determined after 3 days in darkness. Bars indicate SE (n = 4). Only those SE larger than symbol size are shown.
senescence is linked to an increase in MDA level (Figure 4).
It is generally considered that lipid peroxidation is induced by free radicals [5]. If it is the case in the jasmonate-induced lipid peroxidation, then we would expect a reduction of MJ-increased MDA level and a corresponding inhibition of MJ-promoted senescence
Figure 3. Effect of calcium chloride on chlorophyll, protein and
MDA levels in detached rice leaves treated with MJ. Chlorophyll, protein and MDA were determined after 3 days in darkness. The concentration of MJ and calcium chloride were 45M and 10 mM,
respectively. Bars indicate SE (n = 4).
in detached rice leaves by the addition of free radi-cal scavengers such as GSH and SB [14, 21]. This is essentially what we see in Table 1.
20
Figure 4. Effect of linoleic acid (18:2) and linolenic acid (18:3) on
chlorophyll, protein and MDA Levels in detached rice leaves. All treatments included 0.1% Tween 20. Chlorophyll, protein and MDA levels were determined after 4 days in darkness. The concentration of 18:2 and 18:3 was 5 mM. Bars indicate SE (n = 4).
Superoxide can serve as a source to generate more active hydroxyl radicals by Haber-Weiss and Fenton reaction [13, 15]. Transition metals, such as iron and copper, are able to accelerate Haber-Weiss and Fenton reaction [6]. We do not know whether MJ-promoted
Table 1. Effects of free radical scavengers and BP on chlorophyll,
protein and MDA levels in detached rice leaves treated with MJ. Chlorophyll, protein and MDA levels were determined after 3 days in darkness. The concentrations of MJ, GSH, SB, and BP were 45M, 30 mM, 10 mM and 1 mM, respectively. The data
represent mean valuesSE (n = 4)
Treatment Protein Chlorophyll MDA
(mg g 1) (mg g 1) (nmol g 1) H2O 45.340.83 3.710.14 24.333.08 MJ 13.060.83 0.740.08 84.211.32 MJ + GSH 18.480.24 1.680.05 34.832.90 MJ + SB 21.470.79 1.760.14 25.140.91 H2O 43.670.22 4.320.03 MJ 18.910.60 0.700.02 MJ + BP 56.852.07 4.400.02
senescence of detached rice leaves requires metal ions. We investigated this by using BP, a metal chelator. BP was found to be able to inhibit MJ-promoted senes-cence of detached rice leaves (Table 1).
In conclusion, the result of the present investigation suggest that MJ-promoted senescence is a consequence of free radical-induced lipid peroxidation.
Acknowledgements
This work was supported by the National Science Council of the Republic of China (NSC 85-2321-B002-091). This paper is part XXXVIII of the series Senes-cence of Rice Leaves.
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