Light-requiring acifluorfen action in the absence of bulk photosynthetic pigments

PESTICIDE

BIOCHEMISTRY

AND

Light-Requiring VICTORGABA,~NOA

PHYSIOLOGY

31, 1-12 (1988)

Acifluorfen Action in the Absence of Bulk Photosynthetic Pigments COHEN,YOSEPH SHAALTIEL,~AMMI BEN-AMOTZ,* JONATHAN GRESSEL~

Departments of Plant Genetics and *Biochemistry,

AND

Weizmann Institute of Science. Rehovot IL-76100, Israel

Received October 20, 1987; accepted January 27, 1988 The nitrodiphenyl ether herbicide acifluorfen requires light for phytotoxicity even though it alone cannot absorb light. All possible major pigment systems have been implicated as the photoreceptor. We tested whether carotenoids and chlorophyll are essential for phytotoxicity. We used green-photosynthetic (mixotrophic) tomato cell cultures, etiolated cells of the same line (containing carotenoids but no chlorophyll), and carotenoid-free white cells (by continuously culturing etiolated cells on norflurazon). All three cell culture types parallel plants insofar as the first measureable effect is membrane lipoxidation. Acifluorfen at 1 (LM had little effect in darkness, but strongly inhibited growth of all cultures in 40 umol mm2 set- ’ white light. Acifluorfen at 0. I PM did not affect green cells in light, but inhibited the growth of white and etiolated cells. Action spectroscopy showed that 350-nm light was the most effective wavelength inhibiting the growth of white cells with 1 PM acifluorfen, followed by SO-nm, 450-nm, cool-white fluorescent, and 630~nm light, with only a threefold difference between 350-nm and red light. Far-red light was ineffective. These data demonstrate that in this system, chlorophyll, carotenes, cryptochrome, flavins, and phytochrome cannot be the sole photoreceptor for acifluorfen action. Our data, along with all other published findings are consistent with two hypotheses: (a) that acifluorfen interacts with other moieties to produce broad-spectrum chromophore(s) that react(s) with oxygen, forming active-oxygen species in the light: (b) that acifluorfen stimulates the accumulation of chromophoric photodynamic molecules. 0 1988 Academic Press, Inc.

INTRODUCTION

(2, 7), and preloaded fluorescein (9) from treated plants or cells. This action always required both light and oxygen. Lipid peroxidation is commonly caused by herbicides that interact with photosystem II (IO), as well as photosystem I (11). It is not clear which active oxygen species perform the lipoxidation, although there is some indirect evidence for superoxide involvement (12-14). Copper chelators that inhibit superoxide dismutase and ascorbate peroxidase synergize nitrodiphenyl ether herbicide action (15). Conversely, copper penicillamine, a potent quencher of superoxide, protected against paraquat damage (1 l), but only delayed and partially suppressed acifluorfen action (14), possibly due to the continual generation of superoxide by acifluorfen or its product, saturating the quencher, whereas activated paraquat can be sequestered. There is a rapid tissue depletion of ascorbate and reduced glutathi-

There are many diphenyl ether herbicides, including some (e.g. diclofopmethyl) with action similar to phenoxy herbicides. Most nitrodiphenyl ethers require light for action, but even can have diverse modes of action (cf. 1). The discussion below is restricted to one subgroup, the nitrodiphenyl ethers such as acifluorfen and oxyfluorfen as well as chemically unrelated compounds such as lutidine (2) and Nphenyl imide (3) derivatives which seem to produce similar visual symptoms and cause membrane lipoxidation in the light. Acifluorfen-type induced membrane lipid peroxidation has been measured as the release of ethane (3-9), malonyl dialdehyde ’ Present address: Plant Biotech Industries, Milouda, D. N. Ashrat, IL-25201, Israel. ’ Present address: Department of Biochemistry, University of California, Berkeley, CA 94720. 3 To whom reprint requests should be addressed. 1

0048-3575188 $3.00 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

2

GABA

one (6, 16) with a concurrent increase in glutathione reductase (8) after acifluorfen application. Species with high levels of ascorbate were more tolerant to acifluorfen if the levels of a-tocopherol were also high (4). Exogenously added cY-tocopherol partially protected against diphenyl ether action (14). Diphenyl ether damage was partially prevented in some cases by phenylurea herbicides such as monuron and diuron which block photosynthetic photosystem II (2, 5, 11, 17). This was interpreted as a direct requirement of electron transport for diphenyl ether action. The preventative effect of photosystem II inhibiting herbicides could equally be due to a need for the 0, generated by photosystem II for diphenyl ether damage. The protection by photosystem 11 inhibiting herbicides is not universally found (14), supporting the above hypothesis. Physiological levels of diphenyl ethers do not greatly inhibit photosynthetic processes (5, 1S), nor do they cause lipid peroxidation (11) in isolated thylakoids. Acifluorfen rapidly penetrates into intact isolated chloroplasts, even in the dark (19), and then inhibits photosynthesis (18). This difference between thylakoids and plastids was hypothetically attributed to plastid envelopes. Diphenyl ethers could also interact with stromal enzymes or other compounds prior to an interaction with pigment systems. Some nitrodiphenyl ethers (e.g., 20 FM nitrofluorfen) seem to act directly near cyt f in isolated thylakoids (20). Nearly two orders of magnitude higher concentrations of other nitrodiphenyl ethers are needed to achieve this effect (1). The direct involvement of chlorophyll in acifluorfen action at physiological levels has been discounted by many experimental findings. Chlorophyll-free, yellow mutants of rice, soy, and maize were affected by diphenyl ethers, but albino mutants were not (21, 22). Far-red light grown cucumber plants that were incapable of i4C02 fixa-

ET AL.

tion, were sensitive to acifluorfen damage of other photosynthesis pathways, in a light dependent process (23). Etiolated tissue is also acifluorfen sensitive (2, 9). Whereas light is required for acifluorfen action as a herbicide, acifluorfen can also have “dark” effects. Acifluorfen inhibited mitochrondrial respiration in the dark (24) and augmented the dark amplification pathway leading to sporulation of the fungus Trichoderma (25). Indirect data indicate mitochondrial involvement, but only after the tissue with acifluorfen had been “photoactivated” (14). The mode of action of the diphenyl ethers would be clearer if the chromophore required for herbicidal action were known. These herbicides have no inherent absorption in the visible range where they act. The results using chlorophyll-free mutants and far-red grown plants might suggest a role for carotenes. Unfortunately, the albino mutants were not characterized to ascertain at what step pigment synthesis was blocked. Experiments with broadband light sources suggested that most blue light was active (26), further implicating carotenes. The participation of blue light was supported by experiments in which fluridone or norflurazon were used to deplete tissue of carotenoids, and decrease sensitivity to diphenyl ethers (2, 14, 27). It is now known that carotene-free mutants, and norflurazon-treated normal tissue can be pleiotropically missing a large series of other biochemical pathways (28). Conversely, soybean cells depleted of carotenes by norflurazon were as susceptible to acifluorfeninduced photodamage as untreated cells (9), and barley mutants specifically lacking either photosystem 1 or II were still susceptible to a novel nitrodiphenyl ether herbicide (29). It was hypothesized that diphenyl ethers might interact with the blue light photomorphogenic pigment(s) “cryptochrome,” modulating many responses to blue light (3). Indeed, acifluorfen-treated maize seed-

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lings were more phototropic than controls. Recent findings with cryptochromemodulated, photoinduced Trichoderma sporulation cast some double on interpretations of this phenomenon. Acifluorfen increased sporulation, both when applied in the dark just after light and before the inductive light pulse (25). Experiments using monochromatic light, but not at equal energies, showed that red light (and to some extent green light at 550 nm and UV-A at 370 nm) was also effective in promoting acifluorfen-induced cell death in C~lamydomonas (31). Red light was also effective in causing acifluorfen damage in cucumber (23) and could facilitate damage by the lutidine derivative with acifluorfenlike physiological properties (2). These studies rule out the possibility of carotenes acting as the sole photoacceptor for acifluorfen action. The various views about the physiological mode of action and killing and the seemingly conflicting data on photoacceptors suggested a need for an assessment in a simple higher plant system. We therefore reexamined the relationship between the bulk photosynthetic pigments and the effect of acifluorfen on the growth of photomixotrophic, pigment-free cells in culture. The green culture used is capable of low levels of photosynthetic 14C0, fixation and is killed by photosystem II inhibiting herbicides (32). The pigment composition of tomato cell cultures is easily modulated. Cells grown in continuous white light are green. Cells grown in continuous darkness (etiolated) are yellow and contain carotenes and do not “green up” for at least 7 days on transfer to continuous white light. The growth of etiolated cultures is not blocked by photosystem II inhibiting herbicides (32). Etiolated cells depleted of carotenes by treatment with norflurazon appear white. In this paper we report that acifluorfen in the presence of light can both cause lipoxidation and inhibit growth in the absence of both carotenes and chlorophyll

TO

ACIFLUORFEN

DAMAGE

3

and that this effect of acifluorfen can be induced using all wavelengths of light tested between 350 and 630 nm, but not using far-red light. MATERIALS

AND

METHODS

Plant Material

Tomato (Lycopersicon esculentum Mill cv. San Marzano) tissue cultures were isolated and grown on Gamborg’s B-5 medium with 0.5 mg liter- ’ naphthaleneacetic acid, 0.1 mg liter-’ kinetin, 2% sucrose, and 1% agar as previously described (33). This culture has been subcultured as callus for over 12 years and turns green in the light. Green suspension cultures were prepared in the same medium without agar and were kept in the light (30 p.mol m-’ sect i, PAR) at 25°C on a rotary shaker. Etiolated (yellow) cultures were obtained by wrapping the Erlenmeyer flasks with two layers of thick aluminum foil. Carotenoid-free (white) cultures were obtained by continuously culturing (aluminum foil wrapped) etiolated cultures on 1 PM nofflurazon (for more than 12 weekly transfers). They were transferred to 10 FM norflurazon for two weekly transfers before any given experiment. Experimental

Procedures

All experiments were performed with diluted exponentially growing cells, 1 week after the previous transfer. Such cells continue growing exponentially with a minimal lag. All cultures were filtered through sterile 750~km mesh Nitex monofilament nylon cloth to remove large clumps 1 day before experiments. The cultures were diluted to about 10% packed volumejust prior to each experiment. Growth

Measurements

Two methods were used for liquid culture: shake flasks and thin cell layers in dishes. As will be shown under Results, the growth kinetics were the same using both methods.

4

GABA ET AL.

Method A. Initially, experiments were performed in replicate loo-ml Erlenmeyer flasks with 30 ml medium. Packed volume was measured without removing samples, using a sedimentation method (34). Method B. The large numbers of treatments, replicates, and data points in each experiment precluded performing large experiments with Erlenmeyer flasks containing large volumes. It is also hard to uniformly illuminate many flasks. We found that there was sufficient aeration of thin layers of cell suspensions in petri dishes to permit normal growth. Such cultures grew at the same rates as shake flasks (see Results). Thus, 5-ml aliquots containing 10% packed cell volume were pipetted with chemical additions into 60-mm-diameter disposable plastic petri dishes and sealed with Parafilm. Four replicates (minimal) were used per experimental point. At the end of an experimental period, the cultures were washed (with added water) into graduated, tapered, H-ml tubes and centrifuged for 5 min at IOOOg, and the packed volume was read. Light Sources

The light sources used are described in Table 1. White light was provided by cool-

white fluorescent tubes (Osram). Spectral photon distributions and fluence rates were initially measured with a Li-Cor 1800 spectroradiometer and subsequently fluence rates were measured with a Li-Cor 188B photon-flux light meter. All light sources using fluorescent tubes produced some farred light, but with no apparent physiological effect (see Results). All transfers of dark-grown cultures were performed in a darkroom using a dim green safelight made with yellow and blue acylic sheets. Cultures (Method B) were continuously illuminated from above at the fluence rates stated under Results by modulating the fluence rates above the petri dishes with layers of gauze as neutral density filters. Chlorophyll

Cells were rinsed with fresh medium and centrifuged, chlorophyll was extracted with 4 vol of 100% acetone, and the tubes were stored overnight in the dark. Chlorophylls were determined according to Arnon (36) after centrifugation. HPLC Carotenoid Analysis

Pigments were extracted from Buchner filtered tissue with 4 vol of acetone overnight at 5°C. The tissue sediment was re-

TABLE Light

Waveband

Sources

I and Quality

Light source

Red Green

Cool white fluorescent Cool white fluorescent

Blue”

“Daylight” + “tropical daylight” fluorescent

UV-A Far red’

Rayonet RPR tubeb Tungsten halogen spot lamp

Filter

Amax (mm)

Half power bandwidth

(nm)

I Layer red celluloid I Layer each orange and green celluloid Blue plastic

620 550

65 44

460

52

None Wratten 87B + KG-3 Schott heat absorbing glass and a 180-mm water filter

350 720

38 60

tubes

0 Blue acrylic plastic as in Ref. (25). b Southern New England Ultra Violet Co.. Hamden, CT. c Far red as in Ref. (35).

PHOTOSYNTHETIC

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moved by filtration. The pigments were transferred to hexane by addition of water, and the hexane was removed by flash evaporation in the dark. The pigments were redissolved in methylene chloride and identified by HPLC as previously described (37). All extractions were in glass, and Nz was bubbled through all solutions to remove OZ. Enzyme Assay Cell suspensions were broken in a Kontes conical glass-glass homogenizer and total levels of glutathione reductase were immunochemically measured on gels (Western blots), as previously described (38). Membrane

Deterioration

Membrane deterioration was measured by culturing suspensions of the three cell types in duplicate flasks with and without 1 FM acifluorfen. In order to obtain larger samples, and to allow for collection of gases, Corning 25cm’ flat surface tissue culture flasks were used with a hole drilled in the stopper and a sterile septum placed inside the stopper giving a hermetic seal and allowing gas sample removal. Each flask contained 50 ml of 10% packed volume of cells, leaving 26 ml of air space. Because of the greater thickness of cells, the flasks were illuminated with 70-80 kmol m - ’ set- ’ (PAR) fluorescent light while on a rotary shaker at 120 rev min - ’ . Membrane degradation was estimated from the ethane evolved, using a Packard 419 gas chromatograph with a 152 x 3-mm, 120mesh activated-alumina column. Duplicate I-ml samples of the gas phase were removed and injected at intervals. The temperatures were as follows: oven, 30°C; detector, 100°C; injector, 110°C. The nitrogen flow rate was 5 ml mini ’ . This system completely resolved between ethane (evolved with a retention time of 2.60 min) and ethylene (3.32 min). The data are presented as picoliters ethane per milliliter packed volume of cells, after calibrating the system with pure ethane serially diluted in air under atmospheric pressure.

TO ACIFLUORFEN

3

DAMAGE

Chemicals Analytical-grade sodium salt of acifluorfen was kindly provided by Rohm and Haas (Springhouse PA) and analytical-grade norflurazon by Sandoz (Basle, Switzerland). RESULTS

The bulk pigment levels in the suspension cultures at the time of acifluorfen application are presented in Table 2. The levels of carotenes in green and etiolated cells were about the same. The white, norflurazon-treated cells were devoid of measurable carotenoids but were high in phytoene, a carotene precursor. The limit of detection of carotenoids was 2 kg g-’ fresh wt in the white cells, whereas the maximum level in green and etiolated cells was >750 pg gg ’ fresh wt. Acifluorfen had little effect on cell growth in darkness at concentrations as high as 10 uJ4 (Figs. lB, lD, and IF). Higher levels were inhibitory in the dark (data not shown). One micromolar acifluorfen suppressed growth of green, etiolated, and white cultures in light (Figs. lA, lC, and lE), while 0.1 JLM was sufficient to totally suppress the growth of only the etiolated and white cultures. The cells were brown, and in some cases lost volume, showing ‘negative” growth in the cases where acifluorfen completely suppressed growth (cf. Fig. 1A). TABLE

2

Bulk Pigment Levels in Exponentidly Growing Tomato Cultures 7 Days after the Last Transfer

Cell type” Green” Etiolated White’

Total chlorophyll 300 C.5 <5

Carotenes (WLgg-’ fresh wt)

Total phytoenes

900 7.50 (2

15 12 9ocl

a Green cells were grown under 280 kmol mm’ set- ’ (PAR) white light, etiolated and white cells in the dark. b White cells were cultured in the presence of 10 PM norflurazon for two transfers.

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ET AL.

I 0

I 1 Oays after

2 1pM acifluorfen

1

I

3

4

treatment

FIG. 2. Membrane deterioration in the presence of acifluorfen. Ten percent packed volume cell suspensions were treated and gas phase samples were removedfor ethane determination as outlined under Materials and Methods. In contrast to (9), cumulative ethane evolution was measured.

10 7 0

2

4

6

8

IO 0 2 4 6 8 IO 14 TIME (days) FIG. 1. The effect of acifluorfen on growth kinetics of tomato cell suspensions. The pigment contents of the cell types are summarized in Table 2. Growth was measured in shake culture (Method A). These data are the average of three replicates each. Experiments were repeated two or three times. The standard errors of the mean.7 of all points are less than 10% of the means and therefore not drawan.

The death of plants as a result of acifluorfen treatment is rather rapid: usually within a day. It is valid to question whether the same mode of action of acifluorfen is affecting the cells as affects plants. Because the damage to plants is often measured as membrane lipoxidation, we studied this effect of acifluorfen in the cell culture systems. Ethane evolution was used as a measure of membrane lipoxidation (Fig. 2). Acifluorfen increased the level of lipoxidation with a concomitant release of ethane. There was a more than twofold increase in ethane evolution within 2 days after treatment in all treatments indicating membrane damage. It is logical to assume that membrane leakage of water resulting from dam-

age will manifest its physiological effects more rapidly in a leaf surrounded by air than in a cell suspension surrounded by aqueous medium. Because of these data on ethane, it seems valid to assume that the mode of action of acifluorfen is the same in cell cultures and leaves. The maximum light fluence rates used throughout these experiments are about 5% of daylight, so it must be expected that the throes of death will be more prolonged in cell cultures than in a plant in full sunlight. Other reasons for the apparent differences between the cultures and leaves are summarized under Discussion. The higher concentration of acifluorfen (Fig. 1) required to kill green cells, and the apparent delay in ethane evolution in green cells (Fig. 2), may be due to a variety of factors: (a) The green cells may have higher levels of active-oxygen degrading enzymes or oxyradical quenchers which would spare the tissue from the lower concentrations of acifluorfen. (b) Part of the light may be screened by chlorophyll so that either more light or more herbicide is needed. (c) The green cells have higher rates of acifluorfen detoxification. The first possibility

was checked by mea-

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suring the levels of glutathione reductase, a chloroplast enzyme involved in oxyradical degradation that is induced in acifluorfentolerant beans (8). Six isozymes were immunologically detected on the polyacrylamide gel separations of the proteins. There were no reproducible differences in isozyme patterns between the three cell types, and the total enzyme-protein levels were about the same in all three cell types. The green cells were not significantly enriched in any glutathione reductase isozyme compared to the yellow and white cells (data not shown). Still, as green cells photogenerate NADPH, they are better capable of using the existing enzyme to regenerate reduced glutathione than the nonphotosynthesizing cells. The second possibility was studied by measuring the percentage of incident irradiation transmitted through cultures of the various cell types. The etiolated and the white cultures were virtually transparent at the 10% packed volume starting inoculum in all experiments, but the green cultures initially absorbed about 12% of the light, increasing to 33% after the cells had divided two times (Table 3). This could have some screening effect, especially in the chlorophyll-absorbing regions of the spectrum. The possibility of screening was also

Possible

% Packed volume

Screening

TABLE 3 of Putative Acifluorfen by Bulk Pigments

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7

checked by measuring the interaction between different fluence rates of white light with various acifluorfen concentrations (Fig. 3). The green cells in light with very little or no acifluorfen grew somewhat more rapidly than etiolated and white cells in the dark, showing the increment achieved by photosynthesis. The green cells respond to the lowest acifluorfen concentration (0.1 ~44) at the highest fluence rate. It is clear that the suppression of growth is a direct effect of both fluence rate and acifluorfen concentration in all cell cultures. The carotene-free white cells are clearly inhibited at the same fluence rates and acifluorfen concentrations as the etiolated cultures containing more than 350 times more carotene. It is clear from these results that neither chlorophyll nor carotenes directly interact or are indirectly needed with acifluorfen in this cell-culture system. Unlike some sys-

Acceptor

Cell type Green

Etiolated (% Light

10 20 40

TO ACIFLUORFEN

88 86 61

transmitted”) 100 98 98

White

100 98 95

a Light measurements were made just above and beneath cell cultures (Method B) at different packed volumes using the Li-Car 188B light meter with a quantum flux probe and the transmittance was calculated. The 14% incident irradiation absorbed by the petri dish and medium were subtracted to give only transmittance loss due to the cells in these cultures. These cell cultures were not treated with acifluorfen.

WL Ipmol

m-2*-’

)

FIG. 3. Interaction between white lightfluence rate and acifruorfen concentration on suppressing growth of tomato suspension cultures (A) by cell type and(B) by acifluorfen concentration. Cultures were cultir,ated in thinfilms (Method B). These data are the average of three to five experiments with three replicates each. Error bars represent the standard errors of the means.

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terns where acifluorfen was used at 100-300 pM (18, 20, 39), this system requires a minimum amount of herbicide, again suggesting that we are dealing with a primary effect of acifluorfen. The difference between green cultures and white and etiolated cultures are accentuated when the data on the effects of fluence rate (Fig. 3A) are separately replotted for each herbicide concentration (Fig. 3B). It is clear that much more acifluorfen is required to inhibit the growth of the green cells than to inhibit the growth of the white and etiolated cells. The etiolated and white cells have similar responses. It is unlikely that the lack of effect of the lower two acifluorfen concentrations on green cultures can be due to chlorophyll screening. At the higher fluence rates there is far more energy passing through the green cells than is needed to affect the white or etiolated cells, even with screening (Table 3). We used white cells to determine which wavelengths of light are most effective in inhibiting tissue growth in the presence of acifluorfen as the white cells respond to white light as well as the etiolated cells (Fig. 3). White cells have the least bulk pigments (Table 2) and the lowest inherent absorp-

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tion (Table 3) and should give the most accurate data. The data clearly show that the photoresponse resulting in cell death in tomato is due to a nonspecific absorption (Fig. 4A). UV-A (Amax 350 nm) light was somewhat more efficient than green (A,,, 550 nm) and blue (A,,, 460 nm) light which are more efficient than red light (A,,, 630 nm) (Fig. 4B). Far-red light (h,,, 720 nm) at a very high fluence rate (100 kmol rn-?set-‘, 700-800 nm) had no effect. Chlorophyll absorption is very low at 350 nm. Because of the nonspecific action spectrum of acifluorfen photoactivity there seemed to be no reason to follow up this crude action spectrum with a high-resolution action spectrum, as little new information could be gained. DISCUSSION

It is hard to envisage any interaction between the residual carotene (Table 21, acifluorfen, and light, because the same fluence rates affect the carotene-containing and depleted cell types (Figs. 1 and 3). The absorption of light by carotenes in green and red light is infinitely less than in blue light so carotenes cannot be the sole photoreceptor. Any residual chlorophyll in the

z 28100 d 60 f

40

tj o 20 z 2 IO 0 > 0.3 2

I FLUENCE

32 RATE

IO

32

UV

B

G

R

W

(pmol m-2s’l

FIG. 4. Effects of fighI of ~wriou.s wuvelengfhs on ucifluorfen ucrion. (Al The effect of I FM acij7uorfen was measured using different fluewe rates over LI 7-day period. 100 pmol m ’ set ’ far-red light (700-800 nm A,,,,, 720 nm) had no effecr. Error bars represent the standard errors of I/IP means. (B) Action .spectrum of acijluorfen action calculated for 60% inhibition of the growth of the dark control vs quantum effectiveness from (A). Cultures were cultivated in thin films as outlined in Method B. These dara are the average of three experimenls tiith three replicates each.

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etiolated or white tissue would not absorb more in green light than in red or blue light as the data (Fig. 3) indicate. The acifluorfen activity in tissue containing no chlorophyll precludes the sole involvement of chlorophyll. Thus, bulk photosynthetic pigments cannot be directly involved in acifluorfen action in this system. Cryptochrome(s) and flavins do not absorb red light so they too must be precluded as sole photoreceptors. This calls into question whether there is a specific pigment that “photoactivates” acifluorfen in tomato cells, or if indeed acifluorfen is directly photoactivated as an exciplex. The parallelism in the fluenceresponse curves at various wavelengths is consistent with (but not necessarily the result of) a single photodynamic chromophoric species. If there were more than one chromophore, each would have its own fluence-response relationship. If each had a different extinction coefficient, nonparallel dose-response curves would result. Indeed, the general slight decrease in effectivity with increasing wavelength is typical of a brownish pigment with little specific absorption. In all cases where the time course of diphenyl ether action was measured there was always a considerable lag during which there was no effect (2,5,7,8, 14,23,26,27, 40). This could be during a period when the generated active-oxygen species had to overcome the natural defences of the plant. Additionally, this could be a period when diphenyl ethers are being metabolized, conjugating to produce an exciplex or otherwise reacting with a chromophore, or inducing cell components to produce a chromophore. These latter possibilities are consistent with data showing that there was no lag of acifluorfen inhibition in the light if the tissue was pretreated with acifluorfen in the dark. Diphenyl ether action (at physiological concentrations) could not be demonstrated with isolated thylakoids nor with any isolated pigment, but activity has been shown with intact chloroplasts (18). This too suggests that there might be metabolic

TO

ACIFLUORFEN

DAMAGE

9

processing in plastids before herbicidal action. The product(s) of this action could be chromophoric species that may or may not contain a moiety derived from the diphenyl ethers. Such chromophore(s) would have the broad adsorption spectrum typical of diphenyl ether action. Indeed, many metabolites and reaction products of phenolic compounds such as diphenyl ethers are often brownish and have the nonspecific wavelength dependency shown in Figs. 4A and 4B. Severely inhibited cell cultures turned visibly brown within a week. The photoreceptors resulting from diphenyl ether action might vary among species and be different in changing physiological states. This might partially explain the different photobiological data from different laboratories. This would also explain why carotene seems related to acifluorfen action in some cases but not in others (23, 31). Another possibility looms as a feasible reason for the species differences in diphenyl ether action. Acifluorfen can stimulate the biosynthesis of many phenylpropanoid phytoalexin-type compounds (39,40). Such metabolites can be photodynamic (e.g., 41) having the photooxidative activity typical of diphenyl ether damage. Phytoalexins are species-specific, which would give rise to different action spectra. The possibility that photoreceptors are metabolites induced by the herbicide is indirectly supported by the array of structures causing a diphenyl ether-type response. In all systems tested, a lutidine derivative (2. 9) and a N-phenyl imide herbicide (3) caused diphenyl ethertype responses. It is hard to envisage how an enzyme could cleave both diphenyl ethers and these herbicides into a similar photodynamic chromophore. Conversely, inducers of phytoalexins are known to have widely disparate structures. It is clear from our work and others’ previous results that it will be necessary to ascertain what happens to and with diphenyl ether herbicides in plant cells during the lag before photoactivity is apparent. Unfortunately, there are only published reports of

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GABA ET AL.

acifluorfen metabolism in plants that are naturally resistant: the ether bond is cleaved, the halogenated methylphenol group is conjugated to glucose and the nitrophenyl group is conjugated with homoglutathione (42). There have been no publications on what happens to diphenyl ethers in species that are susceptible to their action, even though this would relate to their toxicity. A. Jacobsen (personal communication) has found that almost all acifluorfen remained in the parent form in acifluorfenkilled weeds. This means that acifluorfen was not metabolized to a different form and/or that its interaction with other molecules must be rather loose, e.g., by hydrogen bonding. Another question requiring further study is whether the photoinhibited growth at very low concentrations (Fig. 1 and Refs. (2, 3, 4, 9)) is part of the herbicidal action. The rapid dessication due to membrane lipoxidation causes rapid death in leaves, far more quickly than the response we measured. The rapid effect of acifluorfen in the leaf may be due to an autocatalytic chain reaction due to the initial water loss. There need not be such a cascade in suspension cultures bathed in liquid media. Floating leaf discs also die slowly compared to leaves in air. Such chain reactions would be much greater at the higher fluence rates that a plant “sees” in daylight. In both leaves and suspension cultures there is membrane damage (cf. Fig. 2. and Refs. (29)). The initial water loss in leaves in air should cause stomata1 closure in leaves, causing a lack of COZ. This will cause an increase in oxygen radical production by the leaf photosynthetic apparatus due to the lack of CO, to accept the normally produced reductants. The oxygen radicals will further lipoxidize the cell membranes with further loss of water. Thus, in the leaf in the air, a small amount of membrane leakage is rapidly amplified into dessication. The cell suspensions are bathed in aqueous medium and thus there need not be much damage

due to water loss, especially because the white and etiolated cells lack the photosynthetic apparatus or have only a weak photosynthetic apparatus (green cells). Thus, only secondary effects of acifluorfen action may differ between leaves and plant cells. For these reasons, we believe that in the cells we used, and in leaves of intact plants, the primary site of diphenyl ether action is the same. ACKNOWLEDGMENTS

We thank Dr. N.-H. Chua for the antibody against spinach glutathione reductase and the late Dr. Y. Rudich for use of the spectroradiometer. We acknowledge useful comments on this research from Dr. S. 0. Duke. V.G. held a Royal Society (London)-Israel Academy of Sciences and Humanities Fellowship. J.G. is the Gilbert de Botton Professor of Plant Sciences. Note added in proof. After this manuscript was submitted, the “in press” paper of Matringe and Scalla (43) came to our attention. They demonstrate that diphenyl ether-type herbicides stimulate vast accumulation of tetrapyrroles, which can photodynamically damage cells at the wavelengths we have shown. Their data are thus consistent with our data and predictions and vice versa. They also support the view that the first toxic product is singlet oxygen, as tetrapyrroles are known to produce singlet oxygen in the presence of light and molecular oxygen. REFERENCES

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