Herbicide-induced peroxidation: Influence of light and diuron on protoporphyrin IX formation

PESTICIDE

BIOCHEMISTRY

AND

Herbicide-Induced

BEATE

PHYSIOLOGY

35, 192-201 (1989)

Peroxidation: Protoporphyrin

influence of Light and Diuron on IX Formation’

NICOLAUS,GERHARD SANDMANN,HIROYUKI WATANABE,* Ko WAKABAYASHI,~ AND PETERB~GER

Lehrstuhl f&r Physiologie und Biochemie der Pjlanzen, Universitiit Konstanz, D-7750 Konstanz, Germany; *Research Center, Mitsubishi Kasei Corporation, 1lWO Kamoshida-cho, Midori-ku, Yokohama City, Japan; and fDepartment of Agricultural Chemistry, Faculty of Agriculture, Tamagawa University, Machida-shi, Tokyo 194, Japan February 15, 1989; accepted July 12, 1989 Formation of ethane and protoporphyrin IX as indicators of peroxidizing activity of oxyfluorfen and chlorophthalim have been assayed comparing heterotrophic, dark-grown, glucosesupplemented Scenedesmus with autotrophic cells grown in pure mineral medium. Within 4 hr, protoporphyrin is formed in the dark by heterotrophic cells, as observed to a lesser extent with autotrophic cells, provided glucose is added. Under a nitrogen atmosphere very little protoporphyrin is produced, and the uncoupler CCCP strongly decreases porphyrin formation. Under culture conditions for 4 hr in the light, autotrophic cells also form substantial amounts of the porphyrin without glucose added, and in heterotrophic cells the level vs the dark sample is at least doubled. Diuron inhibits formation of protoporphyrin IX in illuminated autotrophic cells and prevents formation of the light-induced additional porphyrin in heterotrophic cells. Accordingly, under air, diuron completely inhibits peroxidation in autotrophic but not in heterotrophic cells. Using nitrogen or oxygen atmosphere, it can be shown that the peroxidation-inhibiting effect of diuron is not due to depletion of oxygen but to impaired photosynthesis. Photosynthesis (autotrophic cells) or metabolism of external carbohydrate (heterotrophic cells) ensure formation of protoporphyrin IX. Although autotrophic and heterotrophic cells attain different levels of protoporphyrin IX, induced by either oxytluorfen or chlorophthalim, the amount of peroxidative ethane formation is found to be similar. Autotrophic cells need comparatively low light intensity (50 p,Em-*set- *) for herbicide-induced peroxidation while heterotrophic ones exhibit maximum peroxidative activity at about 400 pEm-2sec-‘. This cannot be explained by the different photosynthetic capacity of both cell types, but possibly reflects differences in the light-mediated peroxidation process itself. 0 1989 Academic

Press, Inc.

INTRODUCTION

p-Nitrodiphenyl ethers (1,2), certain pyridine analogs (3), or cyclic imides (4, 5) induce degradation of membrane acyl lipids and pigments (6, 7). The essential herbitidal effect is ascribed to a peroxidative mode of action with radicals being formed under the influence of these herbicides (8, 9). Peroxidation can be measured by membrane leakage, resulting in loss of ions and water, decay of 35S-labeled thylakoidi Dedicated to Professor Dr. W. Simonis, Wiirzburg, on the occasion of his 80th birthday.

bound sulfolipid, or evolution of shortchain hydrocarbons (see Refs. (9) and (10) for review). The mechanism of radical formation has not yet been elucidated. Light, oxygen, and photoreceptors are involved. Tetrapyrroles have been shown as photosensitizers (excited at 405 nm), which accumulate under the influence of peroxidizing herbicides ( 1l-l 3). Accordingly, inhibitors of porphyrin biosynthesis protect against peroxidative membrane degradation (11, 12). Protoporphyrinogen oxidase appears to be the enzyme target in tetrapyrrole metabolism (14, 15). 192

0048-3575189 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

HERBICIDE-INDUCED

Dim-on* and other photosynthesis inhibitors prevent peroxidation in autotrophic Scenedesmus, pea leaf discs, in cucumber seedlings or cotyledons (1, 3, 16, 17), although the lack of diuron influence has also been reported (18-20). Impairment of peroxidation by these inhibitors points to a role of photosynthesis as important for peroxidation in autotrophic cells. The action spectra indeed indicate the involvement of chlorophyll in light-induced peroxidative phytotoxicity (4,21). Since inhibitors of mitochondrial respiration and uncouplers of oxidative phosphorylation diminished peroxidative membrane degradation in different organisms (11, 22, 23), an involvement of mitochondrial metabolism has also been evidenced. It should be mentioned that peroxidizing compounds exert phytotoxicity in the dark using heterotrophic Scenedesmus cells (5). It appears that a multifunctional process eventually leads to peroxidative damage. The start of radical formation may differ (10) and physiological or metabolic conditions of the plant are involved (20, 24, 25). This paper reports on peroxidative activity and protoporphyrin formation observed with two types of peroxidizing herbicides subjecting our model plant Scenedesmus to various external conditions, particularly comparing heterotrophically dark-grown cells with autotrophic light-grown ones.

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193

glucose and yeast extract as described (26). Light was measured between 400 and 700 nm by a quantum meter (LiCor Inc., Lincoln, NE). Before the experiments, algae were cultivated for 24 hr, autotrophic cultures started with a density of 1 ~1 packed cell volume (pcv)/ml suspension and the heterotrophic ones with 2 ~1 pcv/ml yielding at harvest about 2 and 34 ~1 pcv/ml suspension, respectively. Incubation procedure. Herbicide treatment was performed either under culture conditions or in closed vials as follows: 2 ml algae suspension (autotrophic cells 2 ~1 pcv/ml, heterotrophic ones 3 p,l pcv/ml unless indicated otherwise) was shaken in a Warburg apparatus for 17 hr at 23°C in gastight 9-ml vials. This incubation time was found necessary to measure maximum ethane evolution. Gas samples were withdrawn by a head-space injector (1) and determined by flame ionization after separation on an alumina column. Ethane down to 1.8 pmol/ml algae suspension could be measured; 10-U% of ethylene are included in the ethane data given. The culture medium of heterotrophically grown cells was removed before incubation and replaced by the heterotrophic medium with the yeast extract omitted. Bicarbonate (5 mM) was added to the autotrophic cells to ensure photosynthesis during incubation. If not indicated otherwise, light incubation was carried out at 450 FErn-*set - * (about 20,000 MATERIALS AND METHODS lux) supplied by tungsten lamps. In Fig. 1, Cultivation. S. acutus was grown in ster- light intensity was changed by different layers of filter paper. The herbicides were disile liquid culture at 22°C under gassing with carbon dioxide-enriched air (4%, v/v). Au- solved in methanol and the solvent was kept below 0.1% (v/v) in the incubation metotrophic cultures were illuminated with white fluorescent light of about 80 dium. When indicated, vials were gassed with nitrogen or oxygen for 15 min before p,Em-*set-’ (6000 lux) and heterotrophic incubation. (dark) cultures were supplemented with Determination of sulfolipid. Cells were grown with [35S]sulfate and after removal of 2 Abbreviations used: Oxyfluorfen, 2-chlorothe labeled sulfate from the culture medium 1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl) and degradation of [35S]sulfoquinovosylbenzene; chlorophthalim, N-(4chlorophenyl)glyceride in the cells was determined by 3,4,5,6-tetrahydrophthalimide; CCCP, carbonylcyanide-m-chlorophenylhydrazone; diuron (DCMU), 3- loss of the label under the influence of the peroxidizing compounds. After a 24-hr in(3,4-dichlorophenyl)-1,1-dimethylmea.

194

2

NICOLAUS

AL.

Determination of protoporphyrin IX. Extraction of protoporphyrin IX was carried out according to Ref. (13) after a 17- or 24hr herbicide treatment as indicated. After washing the extract of 20 ml algae suspension three times with n-hexane, the tetrapyrrole content in the acetone layer was determined by fluorescence spectroscopy with a Hitachi F-2000 instrument. The concentration of protoporphyrin IX was calculated using the excitation wavelength of 405 nm and the emission wavelength of 633 nm compared with an authentic protoporphyrin IX standard. Chemicals and statistics. Oxyfluorfen was supplied by Rohm and Haas (Spring House, PA) and chlorophthalim was supplied by Mitsubishi Kasei Corp. (Yokohama, Japan). Other chemicals, analytical grade, were purchased from Merck AG, Darmstadt, and Sigma Chemicals, Deisenhofen, both in Germany. All data are means generally given with standard error from three to five independent sets of experiments.

I

:. ;

ET

1400

‘, 0 IIE 1000

RESULTS

z f

5 600 s 'j f 200 100

200 300 Light lntenstty

400 MO IpElm’. sl

600

700

FIG. 1. Ethane formation induced by oxyfluorfen (A) and chlorophthalim (B) and oxygen evolution (C) depending on light intensity in autotrophic (0) and heterotrophic (0) Scenedesmus cells. One hundred percent of ethnne formation was calculated from the maximum ethane value measured which equaled 6.39.5 nmol ethanelml packed cell volume (pcv) both in autotrophic and heterotrophic cells. Heterotrophic cells in the dark form only 3&50% of the chlorophyll than autotrophic ones. Photosynthesis based on chlorophyll, instead on packed cell volume, is the same for both culture conditions.

cubation under culture conditions, the lipids were extracted, separated by thin-layer chromatography, and the sulfolipid spot scraped off and counted (27).

1. Light Requirement for Peroxidative Action As documented in Table lA, heterotrophic cells incubated in the dark with oxytluorfen exhibited growth inhibition and the chlorophyll content did not increase. A strong inhibitory effect on chlorophyll biosynthesis has been reported previously (5, 28). The 35S-labeled sulfolipid, a marker for thylakoid degradation, remained constant and no ethane was evolved. In the light, however, the chlorophyll content present in the cells was degraded together with a strong ethane evolution (Table 1B) comparable to that reported for autotrophic cells (5). Also growth was stalled. The 35Slabeled sulfolipid content disappeared under the influence of these herbicides, although its decrease lagged behind chlorophyll degradation. Ethane evolution in relation to light intensity was found markedly different in het-

HERBICIDE-INDUCED

195

PEROXIDATION

TABLE 1 Influence of Oxyjluorfen and Chlorophthalim (Both 0.5 @I) on Growth, Chlorophyll Degradation, Content, and Ethane Formation of Heterotrophic Scenedesmus Cells

Conditions A. Incubation in the dark At start After 17 hr of incubation: Control (+) Oxyfluorfen ( + ) Chlorophthalim B. Incubation in the light At start After 17 hr of incubation: Control ( + ) Oxyfluorfen (+) Chlorophthalim

(1) Cell density (IL1 pcv/mB

(2) Chlorophyll content wm

Sulfolipid

(3) Labeled sulfolipid (x lo3 dpm/200 ml)

(4) Ethane formation (nmoYml pcv)

4.0

19.0 f 2.0

50

-

6.8 f 0.2 5.1 ? 0.3 5.1 f 0.2

33.7 + 4.0 19.9 t 1.5 20.5 k 1.4

49 45 46

n.d. n.d. n.d.

3.0

18.3 f 2.4

59

-

8.2 e 1.0 3.3 + 0.3 3.7 T 0.6

33.7 2 4.0 3.6 2 1.4 3.0 2 1.2

48 21

n.d. 10.0 + 1.8 9.4 + 2.0

Note. The herbicides were added at the start of incubation; milliliters in columns l-3 refer to algae suspension. Data of column 3 are of a typical experiment. To column 1: apparently, some swelling of cells occurs during incubation with the herbicides, real growth is less than indicated by packed cell volume (pcv); n.d., nondetect-

erotrophic cells as compared to autotrophic cells (see Fig. 1A for oxyfhtorfen). Light intensities of 150-200 p,Em-2sec-’ were sufficient to induce maximum ethane evolution in oxyfluorfen-treated autotrophic cells. In contrast, heterotrophic cells required much higher light intensities to produce similar amounts of ethane. For example, at 150 p.Em-2sec-‘, only 50% of the maximum ethane evolution at 450 were detectable. The same Gm -2sec-1 results were obtained in heterotrophic cells with 5 p,M diuron present. Chlorophthalim gave similar data (Fig. 1B). Photosynthetic net oxygen evolution of autotrophic and heterotrophic cells differed with light intensities, particularly above 100 kEm-2sec-1 (Fig. 1C). However, no correlation between O2 production and ethane formation was evident. 2. Photosynthesis Inhibition and Nitrogen Atmosphere

Autotrophic cells exhibited the same ethane evolution under nitrogen as under

air. A 100% (v/v) oxygen atmosphere suppressed ethane formation by 50-60% vs the air control (Table 2; camp. Ref. (30)). Peroxidative ethane formation of autotrophic cells was stopped by photosynthesis inhibitors (see also Ref. (5)). Ethane evolution by heterotrophic cells was little influenced neither by diuron (Fig. 2, open columns) nor by metribuzin (data not shown). Accordingly, decrease of chlorophyll was not stopped by diuron as found with autotrophic cells. Lack of diuron influence was observed when using air as the incubation atmosphere. When air was replaced by nitrogen (hatched columns), the presence of 5 $I4 diuron suppressed ethane evolution of glucose-supplemented hetero-, trophic cells and bleaching of cells was not observed. Ethane formation without diuron present even increased somewhat under nitrogen. Simultaneous measurement of malondialdehyde formation showed a slightly diminished peroxidative activity under these conditions as compared to normal atmosphere (data not shown). To improve the

196

NICOLAUS

ET AL.

TABLE

Inhibition

2

of Ethane Formation by Diuron in Autotrophic

Scenedesmus Cells under Different Oxygen Tensions with Oxyfruorfen or Chlorophthalim Present Ethane (nmol/ml packed cell volume)

Additions

Air atmosphere

1. Control ( + ) diuron 2. Oxytluorfen ( + ) diuron 3. Chlorophthalim ( + ) diuron

Oxygen-saturated atmosphere

Nitrogen

1-0.4-0.6-I 9.2

8.4

1.2

1.2

8.3 1.4

2.9 1.3 -

12.0 0.6

Note. The cells were incubated for 17 hr in a Warburg shaker as described under Materials and Methods. Oxyfluorfen, chlorophthalim, 0.5 FM; diuron 5 PM.

A

-

Oxyfluorfen

-

-

Chlorophtholim

-

I FIG. 2. Ethane formation and chlorophyll content of heterotrophic Scenedesmus cells in the light under air (open columns); nitrogen atmosphere, including 67% (v/v) oxygen (hatched columns); and nitrogen atmosphere plus glucose oxidase and catalase with 2-370 (v/v) oxygen (closed columns) using oxyjluorfen and chlorophthalim (both 0.5 ~J.M)as peroxidizing herbicides. With diuron present in the sample, the content of oxygen after nitrogen flushing was below 2% (v/v). Control cells exhibited growth and chlorophyll formation (bottom, left) during light incubation in vials under air as well as under nitrogen atmosphere. At the start the heterotrophic samples contained 12 kg chlorophyll/ml algae suspension. Diuron, 5 FM. Ears on top of columns indicate the standard error,

HERBICIDE-INDUCED

anaerobic conditions, glucose oxidase (0.15 mg/ml) and catalase (0.6 mg/ml) were added to the incubation medium (29). Protection by diuron was even better (closed columns). Lack of the diuron effect on ethane evolution under air could also be observed with low light intensities (150 p,Em-2sec-‘). 3. Accumulation of Protoporphyrin

IX

Dark-grown, glucose-supplemented (heterotrophic) cells accumulate protoporphy-

Protoporphyrin

No. of experiments

197

PEROXIDATION

rin IX after a short cultivation time in the dark with peroxidizing compounds like oxyfluorfen or chlorophthalim present (Table 3, lines 1 and 2). This dark porphyrin formation, however, requires oxygen since it is not observed under nitrogen atmosphere (line 3). The level of protoporphyrin increased two- to sixfold under moderate illumination either under air or nitrogen atmosphere (camp. lines 1 and 5 and 3 and 8). After a short incubation, autotrophic cells

TABLE 3 IX Content and Ethane Formation in Autotrophic and Heterotrophic after Treatment with 0.5 @I Oxyfluorfen

Conditions for porphyrin formation

A. Incubation in the dark 1. 4 hr, air 2. 24 hr, air 3. 24 hr; nitrogen 4. 4 hr, air; ( + ) diuron B. Incubation in the light 5. 4 hr, air 6. 4 hr, air; ( + ) diuron 7. 24 hr 8. 4 hr; nitrogen 9. 4 hr; nitrogen, ( + ) diuron C. Peroxidation condition 10. 24 hr dark; strong light 11. 24 hr dark, (+) CCCP 12. 24 hr dark; (+) CCCP strong light

Light-induced ethane formation (nmol/ml pcv)

Protoporphyrin IX (nmol/ml pcv) Autotrophic

Scenedesmus Cultures

Heterotrophic

22 * 5 25 -r. 5 22 k 4 -

300 If: 30 250 ‘- 20 30? 7 270 k 30

156 2 7 (23) 38 2 7

568 + 40 (22) 321 2 28

20 + 5 128 f 14 20 f 5

25? 6 200* 18 302 8

15 ? 6

25”

6

15 f 6

40?

2

15 f 6

182

4

Autotrophic

Heterotrophic

8.0 + 0.4 7.8 f 0.4

7.9 + 0.3 10.0 -+ 0.6

8.9 + 0.7

7.0 f 0.4

4.6 * 0.3

2.2 f 0.3

Note. Scenedesmus was pretreated with oxyfluorfen and additions as indicated under culture conditions, except for lines 3, 8, and 9 which are data obtained from samples kept in 60-ml vials under nitrogen atmosphere. Figures in parentheses of line 5 represent data with 0.5 mM 2,4-dioxoheptanoic acid present, applied to the cells 2 hr before illumination. The control values for both cell types (= minus herbicide) were found to be 15-25 nmol protoporphyrin IX/ml pcv after incubation for 1 to 4 hr. During dark cultivation, autotrophic controls neither formed chlorophyll nor exhibited growth. Light intensity of 6000 lux as used in part B was at optimum to induce (with autotrophic cells) or to enhance protoporphyrin IX formation (with heterotrophic cells). The samples of part C were precultured in the dark with oxyfluorfen, SCCP, and subsequently illuminated for 17 hr with strong light (20,000 lux) under conditions of the peroxidation assay (lines 10 and 12). Under these conditions, the levels of protoporphyrin IX formed within a 1- to 4-hr period were found about 50% less than those of part B, lines 5 and 6. The ethane data were obtained as described under Materials and Methods with atmosphere and additions as indicated under “Conditions of protoporphyrin formation.” Diuron, 5 PM; CCCP, 10 PM.

198

NICOLAUS

produced much less protoporphyrin than heterotrophic cells but only in the light (line 5). In the dark, no protoporphyrin formation above the control was observed (line 2). This is consistent with the fact that autotrophic cells do not form chlorophyll in the dark (see Table 4 of Ref. (5)). A longer light treatment (24 hr) decreased the tetrapyrrole almost to the control level, probably due to photooxidation (line 7). With both autotrophic and heterotrophic cells, tetrapyrrole accumulation was already seen after 1 hr, and the maximum level was attained after 4 hr. 4,6-Dioxoheptanoic acid (documented for a 0.5 mM concentration) and gabaculine (2 mM) almost suppressed protoporphyrin formation (line 5, data in parentheses). Most interestingly, diuron had a strong inhibitory effect as well. In autotrophic cells in the light, accumulation of protoporphyrin IX was almost abolished (line 6), while in the dark, diuron had no effect (lines 1 and 4). In light-incubated, glucose-supplemented heterotrophic cells the additional porphyrin formation, which was due to illumination, was not observed with diuron present (lines 1, 5, and 6). This diuron inhibition was obtained under air as well as under nitrogen (lines 8 and 9). Protoporphyrin IX accumulation was low in strong light for 17 hr (20,000 lux; lines 10

ET AL.

and 12), i.e., under peroxidation conditions (see legend of Table 3). Although autotrophic and heterotrophic cells were markedly different in protoporphyrin IX accumulation, ethane evolution was about the same (for a 17- to 24-hr ethane assay period in the light; camp. e.g., line 2 with the ethane data of line 10, right part of Table 3; or lines 7 and 8 for heterotrophic cells). As shown with heterotrophic cells (lines 11 and 12), the uncoupler CCCP substantially decreased both protoporphyrin and light-induced ethane formation to a similar extent. In autotrophic cells, herbicide-induced accumulation of porphyrin in the dark could be achieved by adding glucose to the incubation medium. During 1, 4, and 24 hr, substantial protoporphyrin levels were built up vs the control kept in mineral medium (Table 4). Mannitol did not show this effect. As assayed with the 24-hr samples, the diuron effect on light-induced ethane formation was diminished in glucose- but not in mannitol-supplemented cells. DISCUSSION

Scenedesmus can be grown autotrophitally in the light as well as heterotrophically in the dark. Under both conditions, green cells are formed with a functional photo-

TABLE 4 Influence of Glucose and Mannitol on Protoporphyrin IX Content and Diuron Sensitivity of Light-Induced Ethane Formation in Autotrophically Grown Scenedesmus Cells Treated with Oxyjluorfen in the Dark Protoporphyrin IX (nmol/ml pcv) 1 hr 1. Autotrophic, mineral medium 2. Autotrophic, (+ ) glucose 3. Autotrophic, ( + ) mannitol

Ethane (nmohml pcv)

1 hr

Incubation for: 4hr

24 hr

( - ) Diuron

(+) Diuron

18

20

25

9.2 f 0.1

2.1 f 1.0

41

89

63

8.9 + 1.6

4.2 f 1.0

30

20

15

a.1 2 0.9

1.4 + 0.5

Note. Protoporphyrin IX was determined after a 1-, 4-, and 24hr cultivation period with oxytluorfen (0.5 p&f) present. During a 24-hr incubation period, cell density increased only in sample No. 2. The ethane formation assay was performed with samples taken after the 24-hr dark cultivation and performed under the light conditions as given under Materials and Methods. Glucose, mannitol, 0.5%; diuron, 5 PM.

HERBICIDE-INDUCED

synthetic apparatus. With peroxidizing herbicides present, chlorophyll formation is stalled (as demonstrated with glucosesupplemented cells in the dark, Table lA), while in the light a strong chlorophyll degradation and ethane formation is observed (Table 1B). Both autotrophic and glucosesupplemented heterotrophic cells accumulated substantial amounts of protoporphytin IX after short-term incubation periods with oxyfluorfen or chlorophthalim present. This finding indicates that protoporphyrin IX is the photosensitizer in herbicidal action under both autotrophic and heterotrophic conditions and reflects the results obtained with cotyledons of higher plants (10, 11) or leaves (22) pretreated with peroxidizing herbicides. On the other hand, after long-term incubation periods, necessary for determination of lipid destruction by ethane evolution, no correlation between tetrapyrrole content and peroxidative ethane evolution could be found (Table 3, lines 5, 7, and 10). One explanationwhich is under scrutiny in our laboratory at the moment-is that only the protoporphytin IX accumulated in a short time is responsible for peroxidative activity of the herbicide. The uncoupler (CCCP) strongly decreased ethane as well as protoporphyrin IX formation (Table 3, lines 11 and 12). Also, antimycin diminished phytotoxicity of acifluorfen (11) and mitochondrial involvement has been suggested previously (22, 23). Autotrophic cells are not peroxidized with photosynthesis inhibitors present (Refs. (1) and (5) or Table 2). Diuron had no effect with heterotrophic cells. Inhibition of ethane evolution by diuron, however, could be restored by preventing (carbohydrate supported) respiration under a nitrogen atmosphere but allowing for photosynthesis by illumination (Fig. 2). Accordingly, treatment with diuron under light and nitrogen atmosphere leads to a dramatic reduction of protoporphyrin IX accumulation (Table 3, line 9) with a concurrent suppression of

PEROXIDATION

199

peroxidative ethane formation (Fig. 2). Peroxidation, as measured as ethane formation, is not oxygen limited under this condition as was claimed by Bowyer and collaborators (31) since under nitrogen, ethane formation was found even higher than under air (Fig. 2). Autotrophic cells produce about twice as much oxygen than heterophic ones (Fig. l), although only the first exhibit the diuron effect under normal air conditions. Furthermore, excess oxygen even diminished ethane gas evolution. As reported by Reiter and Burk (30), a partial oxygen pressure of some percentage O2 (v/v) will ensure maximum hydrocarbon formation. Apparently, the small oxygen content of the nitrogen-flushed sample is sufficient for a substantial protoporphyrin formation (Table 3, line 8) and enough for maximum ethane formation (Fig. 2A, hatched columns). In completely autotrophic cells, oxyfluorfen-induced protoporphyrin IX accumulation could be prevented by diuron leading to protection against peroxidative damage (Table 2; Table 3, lines 5 and 6), but the inhibition of ethane evolution by diuron started to disappear after glucose addition (Table 4). A nonmetabolizable compound like mannitol was found ineffective. This finding has also been reported for cucumber cotyledons (20). Glucose obviously substitutes for photosynthesis products necessary to form protoporphyrin IX. Accordingly, in the light, diuron was found inhibitory against protoporphyrin and ethane formation in heterotrophic cells kept under nitrogen atmosphere but not under air (Fig. 2; Table 3, lines 8 and 9). In air, illuminated heterotrophic cells cannot form additional protoporphyrin IX on top of their dark biosynthesis capacity (Table 3, see lines 1, 5, and 6). Nevertheless, diuron exhibits no effect on ethane formation since the dark tetrapyrrole level ensures maximum peroxidation . Both ATP and reduced pyridine nucleotides are necessary for tetrapyrrole biosynthesis at the step of 8aminolevulinic

NICOLAUS

200

acid formation. Conceivably, protoporphytin accumulation requires metabolic activity, which can be ensured by respiratory carbohydrate metabolism dominant in heterotropic or by photosynthesis in autotrophic cells. Therefore, herbicideinduced protoporphyrin IX formation and peroxidation were inhibited by the uncoupler CCCP and in autotrophic cells by diuron. An action spectrum should indicate involvement of chlorophyll as reported (4, 21). We assume that in higher plants autotrophic and heterotrophic features of growth are mixed during the course of development, which explains the contradictory results with respect to the diuron effect in the literature (for refs. see Introduction). In Scenedesmus, however, the autotrophic condition can be strictly separated from the heterotrophic one, again demonstrating this

ET AL.

alga as an excellent model species to investigate the physiology of peroxidation. Figure 3 summarizes the light and diuron effect on formation of protoporphyrin IX and peroxidation. Compared to autotrophic cells, heterotrophically grown Scenedesmus needs much higher light intensities to induce similar ethane evolution (Fig. 1). This is reminiscent of the report of Sato and collaborators (21) about tolerance of white seedlings of a rice mutant to acifluorfen-ethyl under low light intensity which could not be observed with green seedlings. This tolerance was lost under higher light intensities which resembles our resulted obtained with heterotrophic cells. The radicalic starter reactions taking place when protoporphyrin IX is involved are not yet known. Whether singlet oxygen is formed or a reductantsupported protoporphyrin anion, in both cases different reaction constants and activation energies ought to be involved (see Ref. (10) for assumed reactions). This may yield different dose-response curves in autotrophic and heterotrophic cells. The light requirement for peroxidation with autotrophic and heterotrophic cells apparently cannot be explained by the different photosynthesis capacity of both cell types. ACKNOWLEDGMENTS

Protoporph. degradation

PeroxidatIon

FIG. 3. Scheme summarizing the conclusions on formation of protoporphyrin IX and peroxidation with respect to light, photosynthesis inhibition (diuron effect), and anaerobiosis (nitrogen atmosphere). Respiration (breakdown of carbohydrates by respiration; left side) or photosynthesis (right side) provide for reducedpyridine nucleotides, ATP, and the carbon skeleton necessary for the buildup of protoporphyrin IX. In thefirst case, this metabolism can be interrupted by lack of oxygen, in the second one by inhibition of photosynthesis. In addition, oxygen is necessary for formation of protoporphyrin and the peroxidation process. Apparently some percentage of oxygen (v/v) is suflcient for light-induced ethane formation. Inhibition of ethane formation by diuron in autotrophic cells is not due to decreased oxygen supply limiting peroxidation itself.

The authors are grateful to R. Miller and M. Klett for excellent technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. K. J. Kunert and P. Bdger, The bleaching effect of the diphenyl ether oxyfluorfen, Weed Sci. 29, 169 (1981). 2. G. L. Orr and F. D. Hess, Characterization of herbicidal injury by acifluorfen-methyl in excised cucumber (Cucumis sativus L.) cotyledons, Pestic. Biochem. Physiol. 16, 171 (1981). 3. M. Matringe, J. L. Dufour, J. Lherminier, and R. Scalla, Characterization on the mode of action of the experimental herbicide LS 82-556, Pestic. Biochem. Physiol. 26, 1.50(1986). 4. R. Sato, E. Nagano, H. Oshio, K. Karnoshita, and M. Furuya, Wavelength effect on the action of a N-phenylimide S-23142 and a diphenyl ether acifluorfen-ethyl in cotyledons of cucumber

HERBICIDE-INDUCED

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(Cucumis sativus L.) seedlings, Plant Physiol. 85, 1146 (1987). K. Wakabayashi, G. Sandmann, H. Ohta, and P. Bdger, Peroxidizing herbicides: Comparison of dark and light effects, J. Pestic. Sci. 13, 461 (1988). G. Sandmann and P. Bbger, Mode of action of herbicidal bleaching, in “Biochemical Responses Induced by Herbicides” (D. E. Moreland, J. B. St. John, and F. D. Hess, Eds.), ACS Symp. Series 181, p. 111, Amer. Chem. Sot., Washington, DC, 1982. G. Sandmann and P. Bdger, Peroxidative formation of C,-hydrocarbons from an o-4 polyunsaturated fatty acid (16:304) in the alga Bumilleriapsis, Lipids 18, 37 (1983). P. Haworth and F. D. Hess, The generation of singlet oxygen (‘0,) by the nitrodiphenyl ether herbicide oxytluorfen is independent of photosynthesis, Plant Physiol. 86, 672 (1988). K. J. Kunert, G. Sandmann, and P. Biiger, Modes of action of diphenyl ethers, Rev. Weed Sci. 3,

PEROXIDATION

18.

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35 (1987).

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