Paraquat toxicity is reduced by polyamines in rice leaves
Chin Jung Chang & Ching Huei Kao
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China; Fax: 886-02-3620879 (
Author for correspondence)
Received 30 May 1997; accepted in revised form 13 June 1997
Key words: Oryza sativa, paraquat, putrescine, spermidine, spermine
Abstract
The protective effect of polyamines against paraquat (PQ) toxicity of rice (Oryza sativa) leaves was investigated. PQ treatment resulted in a higher putrescine (PUT) and lower spermidine (SPD) and spermine (SPM) levels in rice leaves. Pretreatment with SPD and SPM, which resulted in a 10- and 20-fold increase in endogenous level of SPD and SPM, respectively, reduced PQ toxicity (30%). Limited reduction of PQ toxicity by exogenous SPD and SPM is most likely due to the fact that they are not readily transported in rice leaf cells and localized to those areas along the cut edges of detached rice leaves [4]. PUT pretreatment did not increase endogenous SPD and SPM levels and had no effect on reducing PQ toxicity. It was found that 1,10-phenanthroline, an iron chelator, treatment reduced the toxicity of PQ (35%) and increased the levels of SPD (27%). The results indicate that reduction of PQ toxicity by SPD and SPM is due to increased activities of catalase (18%) and peroxidase (40%).
Abbreviations: APOD = ascorbate peroxidase; CAT = catalase; GR = glutathione reductase; PAT =
1,10-phenanthroline; POD = peroxidase; PQ = paraquat; PUT = putrescine; SOD = superoxide dismutase; SPD = spermidine; SPM = spermine
1. Introduction
Polyamines occur ubiquitously in plants, animals, and prokaryotes, and their role in growth, development and stress metabolism has been actively investigated [8]. It was proposed that polyamines could take part in cellular defence mechanism against oxidative damage through the inhibition of lipid peroxidation [12, 22, 23]. Free radical scavenging properties of polyamines have also been documented [7].
Paraquat (PQ) is a herbicide widely used in agri-culture and has long been known to exert its phyto-toxic effects by catalyzing the transfer of electrons from photosystem I of chloroplast membranes to mole-cular oxygen, producing oxygen radicals that cause lipid peroxidation and membrane damage [2]. It has been shown that PQ toxicity is enhanced by iron and reduced by an iron chelator in mice and E. coli [13, 14]. Recently, Zer et al. [25] also demonstrated that an iron chelator was effective in reducing PQ
tox-icity in pea leaves. Minton et al. [16] have shown that toxicity of PQ for Escherichia coli is increased over 10-fold in strains defective in the biosynthesis of SPD compared to isogenic strains containing SPD. The increased sensitivity of these SPD-deficient mutants to PQ is eliminated by growing in a medium containing SPD or by endogenous supplementation of SPD by the use of a spd E+
D+
plasmid. The protective effect of polyamines against PQ toxicity has not been examined in higher plants. In the present study, we investigated the protective effect of polyamines against PQ toxicity in rice leaves, and we observed that the toxicity of PQ is reduced by SPD and SPM.
2. Materials and methods
Rice (Oryza sativa cv. Taichung Native 1) was cul-tured as previously described [10]. The apical 3-cm segments excised from the third leaves of 12-d-old
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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 the light (40mol m
2s 1).
Chlorophyll was determined according to Win-termans and De Mots [24] after extraction in 96% (v/v) ethanol. 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 supernatants were used for determina-tion of protein by the method of Bradford [1] and for enzymes assays. For polyamine determination, leaf segments were homogenized in 5% (v/v) perchloric acid. Polyamine levels were determined using HPLC after benzoylation as described previously [3].
Peroxidase (POD) activity was measured using a modification of the procedure of MacAdam et al. [15]. Activity was calculated using the extinction coefficient (26.6 mM cm 1at 470 nm) for tetraguaiacol. Catalase (CAT) activity was assayed by measuring the initial rate of disappearance of H2O2 [11]. The decrease in
H2O2was followed as the decline in optical density at
240 nm, and activity was calculated using the extinc-tion coefficient (40 mM 1cm 1at 240 nm) for H
2O2
[11]. Superoxide dismutase (SOD) was determined by a spectrophotometric method based on the inhibition of a superoxide-driven NADH oxidation according to Paoletti et al. [18]. The activity of ascorbate peroxidase (APOD) was determined by monitoring the decrease in optical density at 290 nm as ascorbate was oxidized, as described by Nakano and Asada [17]. Glutathione reductase (GR) was determined following the oxida-tion of NADPH at 340 nm [9].
3. Results
The chlorophyll (Chl) and protein levels in control leaf segments remained unchanged during 24 h of incubation in the light (Figure 1). A decrease in Chl level induced by PQ was observed after 24 h of treat-ment, whereas PQ induced a sharp decline in protein levels. The decrease in protein level caused by PQ was detected within 4 h of application. Clearly, PQ is more effective in decreasing protein level than Chl level.
PQ significantly increased the level of PUT in the leaf segments (Figure 2). In contrast, the SPD levels in treated segments were not significantly different from that in the untreated segments within 12 h of PQ exposure. After 24 h, however, SPD levels in the
Figure 1. Changes in levels of Chl and protein in detached rice leaves
treated with PQ. Detached rice leaves were treated with either water or 25M PQ in the light. Vertical bars represent standard errors (n = 4).
treated segments fell well below those in untreated controls (Figure 2). Apparent differences in SPM lev-els in treated and control segments were not observed following 6 h of PQ treatment; however, PQ treat-ment apparently blocked the large rise observed in SPM levels in control segments between 12 and 24 h.
Detached rice leaves pretreated with PUT, SPD or SPM had higher endogenous levels of PUT, PUT and SPD, and PUT, SPD and SPM, respectively, than those pretreated with water (Figure 3). To test if polyamines could reduce the toxicity of PQ, as judged by the changes in protein levels, detached rice leaves were pretreated with either water or polyamines for 6 h in the dark and then transferred to either water or PQ for 18 h in the light. SPD and SPM, but not PUT, pretreat-ments reduced the toxicity of PQ (Figure 4).
In the present study, we show that PAT, an iron chelator, treatment reduced the toxicity of PQ in
Figure 2. Changes in levels of polyamines in detached rice leaves
treated with PQ. Detached rice leaves were treated with either water or 25M PQ in the light. Vertical bars represent standard errors (n = 4).
detached rice leaves (Figure 5). In order to determine whether the reduction of PQ toxicity caused by PAT was associated with the increase in SPD and SPM levels, polyamine levels in detached rice leaves treated with PAT for 6 h in the dark were measured. PAT treat-ment increased the levels of PUT and SPD, but did not have a significant effect on SPM concentrations (Figure 6).
The results presented above suggest that high levels of SPD and SPM in detached rice leaves may reduce the toxicity of PQ, and we hypothesize that the effects of
Figure 3. Effect of polyamines on levels of polyamines in detached
rice leaves. Detached rice leaves were treated with either water, PUT, SPD or SPM (5 mM) for 6 h in the dark. Vertical bars represent standard errors (n = 4).
SPD and SPM may be mediated through their influence on oxygen detoxifying systems. To test this hypothesis we examined the effects of SPD and SPM on enzyme activities in the Halliwell-Asada pathway, as well as the activities of CAT and POD in detached rice leaves. Although SPD and SPM did not increase the activities of SOD, APOD or GR, they did increase the activities of CAT and POD in detached rice leaves (Figure 7).
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Figure 4. Effect of polyamines on levels of protein in detached rice
leaves treated with PQ. Detached rice leaves were pretreated with water, PUT, SPD or SPM (5 mM) for 6 h in the dark and then were treated with either water or PQ (25M) for 18 h in the light. Vertical bars represent standard errors (n = 4).
Figure 5. Effect of PAT on levels of protein in detached rice leaves
treated with PQ. Detached rice leaves were pretreated with either water or 5 mM PAT for 6 h in the dark and then were treated with either water or 25M PQ for 18 h in the light. Vertical bars represent standard errors (n = 4).
4. Discussion
Data from the present study indicate that PQ toxicity in detached rice leaves may be modulated by SPD and SPM. This conclusion is based on the observations that (a) pretreatment with SPD and SPM, which increased
Figure 6. Effect of PAT on levels of polyamines in detached rice
leaves. Detached rice leaves were treated with either water or 5 mM PAT for 6 h in the dark. Vertical bars represent standard errors (n = 4).
endogenous levels of SPD and/or SPM, resulted in a reduction of PQ toxicity; (b) PUT pretreatment did not increase endogenous SPD and SPM levels and had no effect on reducing PQ toxicity; and (c) PAT-treatment of leaves increased SPD levels and also reduced PQ toxicity. Our results and conclusions, therefore, appear to be consistent with those of Minton et al. [16], who
Figure 7. Effect of SPD and SPM on activities of SOD, APOD, GR,
CAT, and POD in detached rice leaves. Detached rice leaves were treated with water, 5 mM SPD or SPM for 6 h in the dark. Vertical bars represent standard errors (n = 4).
demonstrated that PQ toxicity was increased in E. coli defective in the synthesis of polyamines.
Quantitation of polyamines in the present inves-tigation was done by measuring the total extractable polyamines, which tells us little about the concen-tration of polyamines in the cellular compartment in question. Furthermore, we have shown previously that SPD and SPM are not readily transported in rice leaf cells and localized to those areas along the cut edges of detached rice leaves [4]. All these would explain why the strong 10- to 20-fold increase in endogenous levels of SPD and SPM, respectively, corresponded to just a little reduction of PQ toxicity (Figures 3 and 4).
Plants accumulate polyamines in response to var-ious types of environmental stress [8]. Very little is known about the effect of herbicides on polyamine levels in plants. DiTomaso et al. [6] reported that napropamide, a soil applied amide herbicide, increased PUT level but had little effect on SPD and SPM lev-els in pea roots. The levlev-els of PUT, SPD and SPM in pea leaves were increased by atrazine, a widely used selective herbicide which interacts with photosystem II in higher plants [26]. In the present work, we have shown that PUT levels increased in PQ treated rice leaves, while SPD and SPM levels decreased.
It was previously demonstrated that feeding PUT to cut leaves of barley produced symptoms similiar to those in potassium deficient and salt stressed plants [5, 20]. Recently, we also reported that growth inhi-bition in suspension-cultured rice cells under potas-sium and phosphate deprivation was closely associated with PUT accumulation [19, 21]. Since an increase in endogenous PUT level does not enhance PQ toxicity (Figures 3 and 4), it seems doubtful that PUT accmu-lation is involved in the sequence of events leading to PQ toxicity.
In considering a possible mechanism for the reduc-tion of PQ toxicity by polyamines, we first speculated that SPD and SPM might inhibit PQ uptake from the medium. Since detached rice leaves were pretreated with SPD and SPM followed by treatment of PQ, our findings cannot be explained by direct competi-tion between PQ and SPD or SPM. Nevertheless, we cannot exclude a possible effect of endogenous SPD and SPM on a PQ transport system.
PQ toxicity results from the production of gen peroxide, the superoxide radical and the hydro-xyl radical formed in the chloroplast during photosyn-thesis [2]. Thus the presence of elevated activities of enzymes such as CAT, POD, SOD, APOD and GR, which would modulate the accumulation of such toxic
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products, might reduce the toxicity of PQ. Our results indicated that SPD and SPM treatments resulted in higher activities of both CAT and POD in detached rice leaves (Figure 7), suggesting that the increased activities of CAT and POD by SPD and SPM pretreat-ment may partially contribute to the reduction of PQ toxicity. To defend against oxidants plants also have low-molecular-mass antioxidants such as glutathione and ascorbic acid. Further research is necessary to clarify the relation among polyamines, antioxidants and PQ toxicity.
The protection afforded by polyamines against PQ toxicity has been proposed to involve scavenging of PQ-derived free radicals [16]. This proposal was based mainly on the previously reported free radical-scavenging properties of polyamines [7] and the conse-quent reduction of lipid peroxidation [23]. However, it was found that PQ toxicity in detached rice leaves was reduced by SPD and SPM, but not by PUT, render-ing the postulated free radical-scavengrender-ing properties of polyamines rather questionable. Furthermore, we found that polyamines per se had no effect on lipid peroxidation in detached rice leaves (data not shown).
Acknowledgement
This work was supported financially by the National Science Council of the Republic of China (NSC 84-2321-B002-097).
References
1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 2. Calderbank A (1968) The bipyridylium herbicides. Adv Pest
Cont Res 8: 127–235
3. Chen CT and Kao CH (1991) Senescence of rice leaves XXX. Levels of endogenous polyamines and dark-induced senes-cence of rice leaves. Plant Cell Physiol 32: 935–941 4. Cheng SH and Kao CH (1983) Localized effect of polyamines
on chlorophyll loss. Plant Cell Physiol 24: 1463–1467 5. Coleman RG and Richards FJ (1956) Physiological studies in
plant nutrition. XVIII. Some aspects of nitrogen metabolism in barley and other plants in relation to potassium deficiency. Ann Bot 20: 393–409
6. DiTomaso JM, Rost TL and Ashton FM (1988) Herbicide-induced diamine accumulation in pea roots. The effect of nopropamide on polyamine levels. Plant Cell Physiol 29: 1367–1372
7. Drolet G, Dumbroff EB, Legge RL and Thompson JE (1986) Radical scavenging properties of polyamines. Phytochemistry 25: 367–371
8. Evans RT and Malmberg RL (1989) Do polyamines have roles in plant development? Ann Rev Plant Physiol Plant Mol Biol 40: 235–269
9. Foster JG and Hess JL (1980) Responses of superoxide dismu-tase and glutathione reducdismu-tase activities in cotton leaf tissue exposed to an atmosphere enriched in oxygen. Plant Physiol 66: 482–487
10. Kao CH (1980) Senescence of rice leaves IV. Influence of benzyladenine on chlorophyll degradation. Plant Cell Physiol 21: 1255–1262
11. Kato M and Shimizu S (1987) Chlorophyll metabolism in high-er plants. VII. Chlorophyll degradation in senescing tobacco leaves: phenolic-dependent peroxidative degradation. Can J Bot 65: 729–735
12. Kitada M, Igarashi L, Hirose S and Kitagawa H (1979) Inhi-bition by polyamines of lipid peroxide formation in rat liver microsomes. Biochem Biophys Res Commun 87: 388–394 13. Kohen R and Chevion M (1985) Paraquat toxicity is enhanced
by iron and reduced by desferrioxamine in laboratory mice. Biochem Pharmac 34:1841–1843
14. Korbashi P, Kohen R, Katzhendler J and Chevion M (1986) Iron mediates paraquat toxicity in Escherichia coli. J Biol Chem 261: 12472–12476
15. MacAdam JW, Nelson CJ and Sharp RE (1992) Peroxidase activity in the leaf elongation zone of tall fescue. Plant Physiol 99: 872–878
16. Minton KW, Tabor H and Tabor CW (1990) Paraquat toxicity is increased in Escherichia coli defective in the synthesis of polyamines. Proc Natl Acad Sci USA 87: 2851–2855 17. Nakano Y and Asada K (1981) Hydrogen peroxide is
scav-enged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22: 867–880
18. Paoletti F, Aldinucci D, Mocali A and Capparini A (1986) A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal Biochem 154: 536–541
19. Shih C-Y and Kao CH (1996) Growth inhibition in suspension-cultured rice cells under phosphate deprivation is mediated through putrescine accumulation. Plant Physiol 111: 721–724 20. Strogonov BP, Shevyakova NJ and Kabanov VV (1972) Diamine in plant metabolism under condition of salinization. Sov Plant Physiol 19: 938–943
21. Sung H-I, Liu L-F and Kao CH (1994) Putrescine accumulation is associated with growth inhibition in suspension-cultured rice cells under potassium deficiency. Plant Cell Physiol 35: 313– 316
22. Tadolini B, Cabrini L, Landi L, Varani E and Pasquali P (1984) Polyamine binding to phospholipid vesicles and inhibition of lipid peroxidation. Biochem Biophys Res Commun 122: 550– 555
23. Tadolini B (1988) Polyamine inhibition of lipoperoxidation: The influence of polyamines on iron oxidation in the presence of compounds mimicking phospholipid polar heads. Biochem J 249: 33–36
24. Wintermans JFGM and De Mots A (1965) Spectrophotometric characteristics of chorophylls a and b and their pheophytins in ethanol. Biochim Biophys Acta 109: 448–453
25. Zer H, Peleg I and Chevion M (1994) The protective effect of desferrioxamine on prarquat-treated pea (Pisum sativum). Physiol Plant 92: 437–442
26. Zheleva DI, Alexieva VS and Karanov EM (1993) Influence of atrazine on free and bound polyamine levels in pea leaves. J Plant Physiol 141: 281–285
