Chapter 9
Porphyric Pesticides Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 08/07/18. For personal use only.
Variation in Crop Response to Protoporphyrinogen Oxidase Inhibitors 1
H . Matsumoto, J. J. Lee , and K . Ishizuka Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan
Tolerance of nine plant species to diphenyl ether (DPE) herbicides oxyfluorfen and chlomethoxyfen were tested in vivo. There was considerable variation in tolerance to the herbicides between the species. Although both herbicides cause photodynamic damage as a result of protoporphyrinogen oxidase (Protox) inhibition, resulting in abnormally high levels of protoporphyrin IX (Proto IX) accumulation, there is little information on the reasons for differential interspecific tolerance to the herbicides. We compare uptake, movement and metabolism, Proto IX accumulation in vivo, Protox inhibition in vitro, and activities of antioxidative systems between the species to investigate the physiological basis of differential tolerance to two diphenyl ethers. Our findings suggest that differential tolerance of the species examined in this study is mainly due to differences in rates of herbicides absorption, Proto IX accumulation, and intrinsic antioxidative activity.
Several classes of herbicides, including diphenyl ethers, cyclic imides and oxadiazoles cause light-dependent bleaching in sensitive plants by causing the accumulation of the photodynamic tetrapyrrole, protoporphyrin IX (Proto IX) (1-14). They are also known to inhibit protoporphyrinogen oxidase (Protox), which is the last enzyme of the common branch of the heme- and chlorophyll-synthetic pathways in plants (15-22). It has been suggested that accumulated protoporphyrinogen (Protogen), a substrate of Protox, leaks from the porphyrin-synthesizing plastid and is oxidized to Proto IX spontaneously and/or by an oxidizing activity located in the plasma membrane (20, 23-25). Proto IX is a potent photosensitizer and can generate singlet oxygen in the light (26-29). This active oxygen species is highly toxic to plants because it effectively peroxidizes membrane lipids. Among the Protox inhibitors, the mechanism of action of diphenyl ether (DPE) herbicides have been investigated most intensively. Although DPE herbicides inhibit a 1
Current address: National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan
0097-6156/94/0559-0120$08.00/0 © 1994 American Chemical Society
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vitally important metabolic process, namely chlorophyll biosynthesis, considerable variation in susceptibility to the herbicides exists between plant species. Tolerance of peanut to fluorodifen (30), rice to chlomethoxyfen (31, 32), and soybean to acifluorfen (33) is due to metabolic detoxification of these chemicals. Therefore, interspecific differential metabolism can account for selectivity in some crop species. However, other factors must be involved in the mechanism of DPE tolerance in some species since they are highly tolerant but poorly metabolize the herbicides (34). In this paper, we confirm variation in plant species in response to DPE herbicides and describe the possible factors involved in their differential tolerance. Response of Crop and Weed Seedlings to Oxyfluorfen and Chlomethoxyfen Growth Response. Tolerance of nine plant species including crops and weeds to the DPE herbicides oxyfluorfen [2-chloro-l-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl) benzene] and chlomethoxyfen [2,4-dichloro-1 -(3-methoxy-4-nitrophenoxy)benzene was evaluated The gramineous and dicotyledonous species were grown to the 2-leaf stage and fully expanded cotyledonous stage, respectively, by hydroponic culture in an environmental chamber maintained at 25/20 °C day/night and with a 12-h photoperiod of 450 u E n r s photosynuietic photon density. For the herbicide treatment, whole shoots excluding the roots of the plants were soaked for 2 h in l u M herbicide solution (containing 1 % (v/v) ethanol as a solvent) in darkness. Plants were kept in darkness for 24 h and then transferred to the growth chamber and grown for 6 days. Fresh weight determination (Figure 1) clearly demonstrated differential response of these plant species to oxyfluorfen and chlomethoxyfen. The shoot fresh weight of corn, the most tolerant species tested, was reduced only 31% compared with non-treated control, and that of tomato, the most susceptible one, was reduced 90% at 6 days after treatment with oxyfluorfen. Chlomethoxyfen was less phytotoxic than oxyfluorfen. Rice was completely tolerant to the chlomethoxyfen treatment This herbicide has been used for barnyardgrass and broadleaf weeds control in paddy rice. The order of the tolerance of the plant species was very similar between the herbicides; corn and rice were tolerant, while dicotyledonous plants such as tomato, cabbage, and buckwheat were very susceptible. 2
_1
Ethane Evolution. Ethane evolution has been determined as an index of oxidative damage caused by the photobleaching herbicides (35-38). The hydrocarbon gas is a decomposition product of peroxidized fatty acids in membrane lipids (39, 40). A time course of ethane production from oxyfluorfen-treated shoots was determined by gas chromatography. When the plants were treated with oxyfluorfen, as in the study of growth response, ethane evolution from the shoots of susceptible species occurred immediately after exposure to the light (Figure 2). Furthermore, susceptible species tended to evolve large amounts of the gas. A small amount of ethane was detected from nontreated plants. This suggested that membrane disruption in the susceptible species was initiated immediately after light irradiation. Because ethane evolution was observed faster than loss offreshweight, it was assumed that membrane disruption led to water loss from the susceptible species.
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oxyfluorfen
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chlomethoxyfen
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Figure 1. Effect of oxyfluorfen and chlomethoxyfen on plant growth. (Reproduced with permission from ref. 34. Copyright 1991 Weed Sci. Soc. Japan.)
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Time
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Figure 2. Ethane evolution from shoots of different plant species treated with oxyfluorfen.
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Mechanism of Differential Tolerance between Plant Species The plants tested hi these experiments showed clear differential tolerance to the DPE herbicides. To identify the basis of differential tolerance, uptake and metabolism of the herbicides, Proto IX accumulation, inhibition of Protox, and activities of antioxidative systems were compared. Uptake and Metabolism. Uptake of oxyfluorfen and chlomethoxyfen by shoots was measured by placing shoots in 1 u M ^-oxyfluorfen or C-chlomethoxyfen solution containing 1% ethanol, and periodically sampling. Shoots were rinsed with distilled water, dried, and combusted with a tissue oxidizer. The radioactivities in shoots were determined by a liquid scintillation spectrometry (Figure 3). Uptake of both herbicides increased throughout 2-h soaking periods in all plant species; however, it was more rapid in the dicotyledonous species, especially in tomato, than the gramineae. The order of the amounts of absorbed C in plant shoots during 2 h were similar between the two herbicide treatments and it was negatively correlated with the order of the tolerance determined by the fresh weight. This indicates that differential uptake may account for some of the tolerance seen in some plant species. Autoradiography of the plants following C-labeled herbicide treatment indicated that little translocationfromtreated parts occurred both in shoots and roots (data not shown). We previously investigated metabolism of absorbed C-oxyfluorfen and C chlomethoxyfen in shoots of five plant species by a thin-layer chromatography (34). None of the plants metabolized oxyfluorfen, and over 90% of radioactivity remained as the parent compound after 24 h incubation. Metabolism of chlomethoxyfen in the plants was also very limited except in rice and cucumber in which only 20 and 51 % of the absorbed activity, respectively, remained as the parent compound The main metabolites in rice were desmethyl chlomethoxyfen and unidentified water-soluble compounds. It seems that metabolism has little importance in differential tolerance of these species to oxyfluorfen. However, the greater metabolic degradation of chlomethoxyfen in rice and cucumber possibly contribute to their tolerance to the chemical. It should be noted that radish was the most tolerant to the herbicides among the dicotyledonous species tested in spite of its greater uptake and less metabolism. Other factors must be responsible for the tolerance of this plant. 1
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1 4
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Proto I X Accumulation. Proto IX content in shoots of oxyfluorfen-treated plants was determined. The shoots of the plants were soaked in 1 u M solution for 2 h in darkness. They were then either exposed to light (450 uEm^sec" ) or kept in darkness. Among five species tested, only cucumber and buckwheat showed phytotoxic symptom during 8 h incubation in the light. Porphyrins were extracted from their shoots and analyzed as previously described (11). In the light (Figure 4A), Proto IX content rose rapidly during the 1 h in all plants; however, profiles of Proto IX during the following light exposure varied greatly among the species (41). Buckwheat, one of the most sensitive species, accumulated the greatest amount of Proto IX until 2 h and then it decreased rapidly. Cucumber, also susceptible, showed higher Proto IX accumulation until 1 h. Amounts of Proto IX in untreated control plants were less than the limit of detection (1 nmol / g dry weight) throughout the time course. Therefore, it seems that the amounts of oxyfluorfeninduced Proto IX within a shot period (1-2 h) correlate with tolerance to the herbicide in some plant species. Watanabe et al. (42) also reported that the intensities of cyclic imide1
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PORPHYRIC PESTICIDES
oxyfluorfen
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chlomethoxyfen
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0
Time (min) 14
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Figure 3. Uptake of C-oxyfluorfen and C-chlomethoxyfen from shoots of different plant species. (Reproduced with permission from ref. 34. Copyright 1991 Weed Sci. Soc. Japan.)
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induced bleaching and amount of ethane evolution from Scenedesmus acutus correlated well with Proto IX level after 1 h incubation. The faster* accumulation of Proto IX in buckwheat may be due in part to greater absorption of oxyfluorfen from its shoots. Interestingly, Proto IX content in rice and barnyardgrass increased constantly throughout 8-h light irradiation. Proto IX level in rice at 8 h was much higher than that in the susceptible species, however, no phytotoxic symptoms were observed. Therefore, it is hypothesized that rice has some mechanism to protect it from Proto IX-induced lipid peroxidatioa In contrast, radish accumulated less Proto IX and this may relate to the herbicide tolerance, although the mechanism which suppresses Proto IX accumulation is still unclear. Varying rates of porphyrin synthesis (43) and Protogen oxidizing activity in the plasma membrane (20) have been suggested as mechanisms for the differential Proto IX accumulation. At 8 h, no correlation between amounts of Proto IX in the plants and their tolerance to the herbicide was observed. Interestingly, the decrease of accumulated Proto IX in the light was initiated earlier in oxyfluorfen-susceptible species. Since Proto IX is known to be photolabile (#), photodegradation of Proto IX in oxyfluorfen-sensitive plants may explain rapid dissipation seen in these plants. In the susceptible species, the porphyrin synthesis pathway located in the plastid membrane was apparently disrupted within a shorter period, and this interrupted further accumulation of Proto IX. The susceptible plants may have less capacity to protect the membranes from the active oxygen-induced peroxidation. Little is known about the nature of the photodegradation products of Proto IX and their possible involvement in the photodynamic action of DPE herbicides. In darkness (Figure 4B), no Proto IX accumulation occurred in rice, radish, and cucumber. In contrast, buckwheat and barnyardgrass accumulated significantly large amounts of Proto IX although the accumulation rates were slower than in the light. This indicated that, in some species, light is not necessary for oxyfluorfen-induced Proto IX accumulation. This result is different with that reported by others (44, 45) who found that light is mandatory for Proto IX accumulation. Our data also indicated that light acts as an enhancer of oxyfluorfen-induced Proto IX accumulation in the plants. Again, no Proto IX was detected in non-treated plants. 6-Aminolevulinic acid (ALA) synthesis from glutamate has been reported as a ratelimiting step in the porphyrin synthesis and the step is regulated by light (46, 47). Therefore, there is a possibility that the rate of A L A synthesis and/or its regulation by light differed among the species and could account for some of the tolerances seen in certain plant species. To examine this possibility, the contents of A L A in light and dark and the effects of oxyfluorfen on the contents were compared between three plant species (Figure 5). As in the above mentioned experiments, whole shoots of intact plants were exposed to 1 u M oxyfluorfen solution in darkness and then exposed to the light or kept in darkness. A L A was extracted with 4% TCA and determined spectrophotometrically (48). The data indicated that the rates of A L A synthesis in the plants were somewhat greater in the light; however, the amounts of A L A differed considerably between the species. A L A in buckwheat was significantly higher than the other species and the plant generated a considerable amount of A L A even in darkness. In Euglena gracilis, which has the same pathway of A L A synthesis as higher plants, A L A synthesis enzymes, especially GSA aminotransferase were reported to be light dependent (47). Because buckwheat shows a rather high rate of A L A formation in darkness, it would be a interesting to investigate the light regulation of ALA-forming enzymes. The data also showed that A L A formation was not inhibited or stimulated by oxyfluorfen. Thus, the greater accumulation of Proto IX in
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Figure 4. Time course of protoporphyrin IX accumulation in shoots of differential species in the light (A) and dark (B). (Figure 4A: Reproduced with permission from ref. 41. Copyright Academic Press.)
A : Light
B : Dark
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