Photosynthesis Research 32: 1-10, 1992. © 1992 KluwerAcademic Publishers. Printedin the Netherlands. Regular paper

Physiological effects of sublethal atrazine on barley chloroplast thyl membranes

oid

Winston Raul De la Torre ~ & Kent Oliver Burkey 2

Present address: Horticulture Department, Recinto Universitario De Mayaguez, POB 5000, Mayaguez, Puerto Rico 00709-5000; 2 United States Department of Agriculture, Agricultural Research Service and Departments of Crop Science and Botany, North Carolina State University, Box 7631, Raleigh, NC 27695-7631, USA (Author for correspondence) Received 26 June 1991; accepted in revised form 16 November 1991

Key words:

chlorophyll a/b ratio, chlorophyll-proteins, electron transport, herbicide, photosynthesis

Abstract

This study was conducted to more clearly define the physiological effects of PS II herbicides on chloroplast thylakoid membrane activity and composition. Barley (Hordeum vulgare L. cv Boone) was grown in hydroponic culture at 20 °C in a growth chamber with a light intensity of 500/.Lmole photons m 2 s-1. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine), a Photosystem II herbicide, was supplied continuously via the roots to 7-day-old plants. Atrazine concentrations greater than 0.07 ppm (0.32/xM) were associated with decreased leaf chlorophyll (chl), lowered chl a/b ratio, inhibition of chloroplast electron transport, and plant death within 1 to 2 weeks. Atrazine at 0.07 ppm was defined as sublethal because no toxic effects were observed. Sublethal atrazine induced a decrease in chl a/b ratio with no effect on leaf chl content. Photosynthetic electron transport was either unaffected in fully expanded leaves or slightly stimulated in expanding leaves by treatment of intact plants with 0.07 ppm atrazine. The major effect of sublethal atrazine was on the chl-protein complex composition. Sublethal atrazine increased the level of the Photosystem II light-harvesting complex (LHC-II) and lowered the level of the CPla Photosystem I complex relative to controls. The numbers of Photosystem II and Photosystem I reaction centers and cytochrome b6/f complexes per unit chl were not affected by sublethal atrazine. The overall result was an atrazine-induced redistribution of light-harvesting chl from Photosystem I to Photosystem II with no effect on the number of thylakoid membrane-protein complexes associated with electron transport.

Abbreviations: asc - sodium ascorbate; BQ - 2,5-dimethyl-p-benzoquinone; chl - chlorophyll; cyt cytochrome; DCIP - 2,6-dichlorophenolindophenol; MV - methyl viologen; P-700 - reaction center of Photosystem I; PS I - Photosystem I; PS II - Photosystem II; TMPD - N,N,N,'N'-tetramethyl-pphenylenediamine

Introduction

Plants growing in the presence of sublethal levels of PS II herbicides exhibit certain characteristics similar to plants growing under low light (Fedtke 1982). Altered photosynthesis and carbohydrate

content (Fedtke 1972, 1973),'reduced chl a/b ratio (Fedtke et al. 1977, Kleudgen 1978, Fedtke 1979), and enhanced stacking of chloroplast thylakoid membranes (Lichtenthaler et al. 1982, Mattoo et al. 1984) are typical responses to low levels of PS II herbicides. Effects on chloroplast

2 thylakoid membrane lipids (Mattoo et al. 1984, Grenier et al. 1987) and the turnover of the Dl-protein of Photosystem II (Mattoo et al. 1984) are associated with the sublethal herbicide response. Because reduced chl a/b ratio (Leong and Anderson 1984, De la Torre and Burkey 1990a) and enhanced grana stacking (Lichtenthaler et al. 1982, Davies et al. 1986) are characteristics of low irradiance environments, the physiological effects of PS II herbicides are often described as a shade response. These results suggest that the chemical induction of a low light response may be possible. This study was conducted to more clearly define the effects of PS II herbicides on barley chloroplast thylakoid membranes and compare the results with irradiance effects (De la Torre and Burkey 1990a,b) on the same membrane system.

Materials and methods

Growing conditions Barley (Hordeum vulgare L. cv. Boone) was grown in hydroponic culture. Seeds were germinated in the dark at 30 °C for 4 d in germination paper. Barley seedlings of uniform size were then transplanted into 1 liter beakers (4 plants/ beaker) containing 900ml Hoagland's solution (Hoagland and Arnon 1950) adjusted to pH 5.2 with KOH. The solution contained full strength trace elements, but only half strength of the major metabolic elements. Ammonium nitrate was included in the nutrient solution to give a nitrate/ammonium ratio of four and a total nitrogen concentration of 9 mM. Iron was added as Sequestrene* to a final concentration of 3 mg Fe/liter. The nutrient solution was continuously aerated and replaced on alternate days to prevent fluctuations in pH and nutrient content. Plants were grown at 20°C with a 16h photoperiod. Illumination was provided by a combina* Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture or the North Carolina Agricultural Research Service and does not imply its approval to the exclusion of other products that may also be suitable.

tion of fluorescent and incandescent lamps that generated a light intensity of 500/zmole photons m

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s

-1

Barley seedlings were grown in nutrient solution for 7 d prior to atrazine treatments so that plants consisted of fully expanded primary leaves and rapidly expanding second main shoot leaves. Atrazine was added as a concentrated (10 000 X) atrazine/methanol solution to the nutrient solution at the beginning of day 8 and on subsequent days when the nutrient solution was replaced. Methanol only served as the control treatment.

Chlorophyll determination Chl per leaf area and chl a/b ratios were determined on leaf disks from fresh tissue. Chl was extracted from leaf tissue with dimethylformamide at room temperature in the dark for 15 to 20 h. The chl concentration and chl a/b ratio of leaf tissue and thylakoid membrane extracts were determined spectrophotometrically (Moran 1982).

Thylakoid membrane isolation Primary and second leaf apical segments, 4- and 6-cm respectively, were used to prepare thylakoids on each harvest day according to published procedures (De la Torre and Burkey 1990a). The final membrane pellet was resuspended in cold isolation buffer and stored on ice during electron transport assays. Membrane preparations were then frozen in liquid N 2 and stored at - 8 0 °C for later analysis of components.

Photosynthetic electron transport assays Uncoupled whole chain activity was measured either as DCIP reduction (H20--~DCIP) or as oxygen consumption (HEO--)MV). PSII electron transport activity (H20--~ BQ--~ DCIP) was measured as DCIP reduction in the presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone to block electron flow from the plastoquinone pool to the cyt b6/f complex. PSI activity (asc/ TMPD--~MV) was measured as oxygen consumption in the presence of 3-(3,4-dichlorophenyl)-l,1 dimethyl urea to inhibit PS II. De-

tails have been published elsewhere (De la Torre and Burkey 1990b).

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P S I reaction centers were quantitated by the reversible light-induced P-700 absorbance change at 697nm using an extinction coefficient of 64 mM -1 cm -1 (Himaya and Ke 1972). The cyt br/f complex content was determined from reduced (hydroquinone) minus oxidized (potassium ferricyanide) difference spectra of cyt f in Triton X-100 solubilized thylakoid membranes (100 tzg chl/ml) using an extinction coefficient of 1 8 m M - l c m -1 (Hurt and Hauska 1981). PSII reaction centers were quantitated by the stoichiometric binding of [14C]-atrazine to thylakoid membranes (Tischer and Strotmann 1977). Details for the P-700, cyt f and atrazine-binding assays have been published elsewhere (De la Torre and Burkey 1990b). Mild SDS-PAGE analysis of chl-protein complexes was performed using the procedure of Anderson et al. (1978) as modified by De la Torre and Burkey (1990a).

Results

I. Concentration dependence of atrazine effects

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Atrazine Concentration (ppm) Fig. 1. Effect of atrazine concentration on leaf chl content and chl a / b ratio. Seven-day-old barley seedlings were supplied with atrazine for 7 d followed by pigment analysis of primary leaf tissue. Each point represents the mean -+SD (shown by error bars) of three independent sets of plants.

Leaf chlorophyll content The chl content per unit leaf area was affected by atrazine supplied via the roots to barley plants grown in hydroponic culture. An atrazine concentration of 0.07 ppm appeared to be the transition point between sublethal and lethal effects. At 0.07 ppm, a small decrease in leaf chl was observed in certain experiments (Fig. 1A), but not others (Fig. 5A). Plants maintained at an atrazine concentration of 0.07 ppm did not show toxic effects after 21 d, the longest treatment period examined during this study (data not shown). A concentration of 0.1 ppm resulted in chlorosis and loss of turgor after a 10 d atrazine treatment. Barley supplied with 0.3 ppm atrazine exhibited chlorosis and loss of turgor within 5 to 6d.

Chlorophyll organization Chl a/b was found to be a sensitive parameter to

monitor the response of barley to atrazine. Chl a/b ratio declined with increasing atrazine concentrations when plants were treated for 7 d with the herbicide (Fig. 1B). A small decrease in chl a/b ratio was observed at 0.03 ppm atrazine with the maximum reduction at 0.1 ppm. Higher concentrations of atrazine did not cause additional reductions in chl a/b ratio, probably because herbicide toxicity had completely altered normal metabolic functions. The altered chl a/b ratio suggested that atrazine affected the distribution of chlorophyll between the chl-protein complexes. To characterize the organization of chl, primary leaf thylakoids from control and atrazine treated plants were analyzed by mild SDSPAGE 'green' gels to examine the distribution of chl between chl-protein complexes. Seven chlcontaining bands were resolved (Fig. 2) and

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identified according to the nomenclature of Anderson et al. (1978). The CPla and CP1 chlprotein complexes of PSI were the highest molecular weight bands. The absorbance spectrum of CPla contained a small amount of chl b (data not shown), an indication that CPla consisted of the CP1 reaction center core of PSI associated with the LHC-I light-harvesting complex as reported by Kuang et al. (1984). CPa consisted mainly of PS II chl a complexes, but is known to contain a variable amount of LHC-I under conditions that dissociate CPla into the CP1 and LHC-I components (Metz et al. 1984). LHCP 1, LHCP 2 and LHCP 3 were different molecular weight forms of LHC-II, the major light-harvesting chl-protein complex of PS II. FP is free pigment released from the complexes during the detergent solubilization. Atrazine treatments had no effect on the percentage of total chl associated with the CPa chl-protein of PS II (Fig. 3A). The percentage of total chl associated with P S I chl-protein complexes (CP1 + CPla) declined with increasing concentrations of atrazine (Fig. 3B). A corresponding increase in the percentage of chl associated with LHC-II was observed as the atrazine concentration increased (Fig. 3C).

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Atrazine Concentration (ppm) Fig. 3. Effect of atrazine concentration on chl-protein complex composition. SDS-PAGE was conducted on thylakoid membranes from barley primary leaves subjected to a 7-d atrazine treatment. The peak areas defined in Fig. 2 were integrated by computer to determine the percentage of total chl associated with each chl-protein complex. LHC-II represents the summation of areas for LHCP 1, LHCP 2 and LHCP 3. The free pigment levels were 12 -+ 2% of the total chl. Each point represents the mean -+SD (shown by errors bars) of three independent sets of plants.

Photosynthetic electron transport Chloroplast electron transport measured in vitro was sensitive to the amount of atrazine supplied to intact plants. Atrazine concentrations of 0.07ppm and below had no effect on whole chain electron transport activity (Fig. 4). Inhibi-

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tion of electron transport was observed in barley plants treated for 7 d with atrazine concentrations of 0.1 ppm or greater. The large standard deviation associated with the 0.1 ppm data reflected the transition from sublethal to lethal effects, such that 0.1 ppm caused inhibition after 7 d in some experiments but required slightly longer times in other experiments. Atrazine treatments that inhibited electron transport also caused plant death within 1 to 2 weeks (data not shown).

H. Response of thylakoid membrane activity and composition to sublethal levels of atrazine Chlorophyll organization Experiments were conducted to examine the kinetics of the sublethal atrazine response and provide a more complete analysis of sublethal atrazine effects on thylakoid membrane activity and composition. Based on the atrazine concentration dependence of leaf chl (Fig. 1A) and chloroplast electron transport (Fig. 4), a concentration of 0.07 ppm was selected to study sublethal effects. Treatment of 7-d old plants with 0.07 ppm atrazine had no effect on chl per unit leaf area (Fig. 5A), but resulted in a large decrease in chl a/b ratio (Fig. 5B) that occurred slowly over a 10 d period. Distinct changes in chl-protein complex composition were associated with the reduction in chl a/b ratio. Sublethal

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Fig. 5. Sublethal atrazine effects on leaf chl and cht a/b ratio. Seven-day old barley seedlings were treated with 0.07 ppm atrazine and changes in primary leaf pigments analyzed over time. Control (O) and 0.07 ppm atrazine (O) treatments. Each point represents the mean +-SD (shown by error bars) of three independent sets of plants.

atrazine had no measurable effect on the percentage of total chl associated with the CPa complex of PS II (Fig. 6A). A decrease in PS I complexes (CP1 + CPla) was observed after a 7-d treatment with 0.07 ppm atrazine (Fig. 6B) and was correlated with an increase in the percentage of total chl in the LHC-II complexes (Fig. 6C). A close inspection of the P S I chl-protein complex data revealed that atrazine affected C P l a , not CP1 (Fig. 7).

Photosynthetic electron transport Sublethal atrazine did not affect uncoupled whole chain photosynthetic electron transport (Fig. 8A, Table 1) or the P S I and PS II specific partial reactions (Table 1) in primary leaves where leaf expansion was complete prior to the atrazine treatment. For second main shoot leaves

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Fig. 7. Effect of sublethal atrazine on PSI chl-protein complexes in barley. Thylakoid membranes from control (O) and 0.07 ppm atrazine (@) treatments were isolated from primary leaves and analyzed by mild SDS-PAGE. Upper panel, percent chl associated with the CP1 complex. Lower panel, percent chl associated with CPla. Each point represents the mean +SD (shown by error bars) of three independent sets of plants.

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Fig. 6. Effect of sublethal atrazine on chl-protein complex composition in barley. Thylakoid membranes from control (©) and 0.07 ppm atrazine (@) treatments were isolated from primary leaves and analyzed by mild SDS-PAGE. (A) Percent chl associated with the CPa complex of PSII. (B) Percent chl associated with PSI complexes (CP1 + CPla). (C) Percent chl associated with LHC-II. Each point represents the mean -+SD (shown by error bars) of three independent sets of plants.

u n d e r g o i n g expansion during the t r e a t m e n t p e r i o d , w h o l e chain electron transport (Fig. 8B, T a b l e 1) and P S I and PS II reactions (Table 1) w e r e stimulated by the presence of 0 . 0 7 p p m atrazine. I n o t h e r experiments, p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t was elevated in the third,

f o u r t h , fifth and sixth main shoot leaves when barley was g r o w n in the presence of 0.07 p p m atrazine for 21 d (data not shown). T r e a t m e n t of plants with 0.07 p p m atrazine had no effect on the n u m b e r of P S I or PS II reaction centers or cyt b 6 / f c o m p l e x e s in chloroplast thylakoid m e m b r a n e s (Table 2).

Discussion

T h e study of physiological effects of herbicides requires criteria to distinguish lethal f r o m sublethal effects. A n u m b e r of factors will influence the definition of sublethal. Plants metabolize herbicides at different rates so that the effective c o n c e n t r a t i o n for induction of a physiological r e s p o n s e must be d e t e r m i n e d for each com-

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TimeAfterAtrazineApplication(Days) Fig. 8. Effect of sublethal atrazine on photosynthetic electron transport in barley. Whole chain activity (H20 ~ DCIP) was measured in thylakoid membranes from control (O) and 0 . 0 7 p p m atrazine (@) treatments. (A) Primary leaf. (B) Second main shoot leaf. Each point represents the mean +-SD (shown by error bars) of three independent sets of plants.

pound/species combination. Culture method will also be a factor. For example, hydroponic culture at a constant herbicide concentration is a different protocol from a single application of herbicide to roots in soil. Therefore, sublethal must be empirically determined for each experimental system. For barley grown in hydroponic culture, sublethal was defined as 0.07 ppm atrazine because this concentration was the maximum amount of herbicide that could be supplied continuously without phytotoxic symptoms. At 0.07ppm (0.32/~M), atrazine altered chl a/b ratio (Figs. 1B and 5B) without significantly affecting leaf chl (Figs. 1A and 5A) or photosynthetic electron transport (Fig. 4). This compares with sublethal atrazine concentrations of 0.5/~M atrazine for Spirodela (Mattoo et al. 1984) and 1.15/zM atrazine for Lemna (Grenier et al. 1987) grown in hydroponic culture. A single application of herbicide at a much higher concentration has been used for plants growing in vermiculite or sand (Fedtke 1972, Fedtke et al. 1977, Kleudgen 1978, Fedtke 1979). The decrease in chl a/b ratio following treatment of barley with sublethal atrazine (Figs. 1B and 5B) has been commonly observed in plants treated with PS II herbicides (Fedtke et al. 1977, Kleudgen 1978, Fedtke 1979, Lichtenthaler et al. 1980), although lethal and sublethal conditions were not always clearly distinguished in these

Table 1. Effect of atrazine on photosynthetic electron transport activity

Primary leaf Control

Second leaf 0.07 ppm

Control

0.07 ppm

Whole chain (H20--+ DCIP) (/zmoles DCIP mgchl i h-Z)

447 -+ 50

459 +_ 18 (2)

454 _+ 34

550 -+ 8 (21)

Whole chain (H20--+ MV) (/* moles 02 mg chl l h - l )

249 --- 24

250 _+ 15 (0)

238 _+ 27

301 -+ 10 (26)

92-+4

100+_3(8)

88+- 2

1421 -+ 122

1400 _+ 40 (0)

PS II ( H 2 0 - + B Q - + DCIP ) ( / x m o l e s D C I P m g c h l 1 h ~) PS I (asc / TMPD ---, MV) (/1 moles O z mg chl- 1 h 1 )

1543 - 83

102-+3(16)

1762 -+ 60 (14)

Each number represents the mean ---SD of three independent sets of plants harvested after a 7-d treatment with 0.07ppm atrazine. The numbers in parenthesis represent the percent increase relative to the respective control.

8 Table 2. Effect of atrazine on photosynthetic electron transport components Primary leaf

Second leaf

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0.07 ppm

Control

0.07 ppm

P-700 (mmoles mol chl- 1)

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2.8 + 0.3

Cytochrome f (mmoles mol ch1-1 )

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2.3 -+ 0.1

2.4 --- 0.2

Atrazine binding protein (mmoles mol chl- 1)

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3.8 --- 1.0

Each number represents the mean +SD of three independent sets of plants harvested after a 7-d treatment with 0.07ppm atrazine.

earlier studies. Differences in chl a/b ratio are known to be related to differences in the distribution of chl between the chl-protein complexes of PS I and PS II (Leong and Anderson 1984, De la Torre and Burkey 1990a). In barley, the atrazine-induced decrease in chl a/b ratio reflected a decrease in P S I chl-protein complexes (Fig. 6B) and an increase in the LHC-II lightharvesting complex of PS II (Fig. 6C). The decrease in P S I complexes was exclusively associated with a reduction in CPla, with no differences observed in CP1 between atrazine and control membranes (Fig. 7). Based on the following argument, the reduction in CPla can be interpreted as a reduction in the LHC-I lightharvesting complex. First, CPla is known to consist of LHC-I associated with the CP1 reaction center core and several additional low molecular weight polypeptides (Kuang et al. 1984). Second, the number of PSI reaction centers was not affected by atrazine because P-700 content was not affected by the herbicide (Table 2). Thus, the reduction of chl in CPla represented a loss of LHC-I. The decline in LHC-I and the increase in LHC-II resulted in a shift in light-harvesting capacity from PSI to PS II in response to atrazine, presumably mediated by the degradation of LHC-I and the synthesis of additional LHC-II units. Apparently, regulatory mechanisms within the plant compensate for sublethal atrazine effects on PSII by increasing PSII antenna size and reducing PSI lightharvesting components. Perhaps, this regulation is linked to the metabolism of the Dl-protein of PS II (Mattoo et al. 1984).

The atrazine effects on chl-protein complex composition in barley were similar to bentazon treatment of radish (Lichtenthaler et al. 1982), but are in contrast with Lemna minor (Grenier et al. 1987). An atrazine effect on Lemna chlprotein composition may have been obscured by the 18 to 20 percent free chl released from Lemna thylakoids during detergent solubilization. Our mild SDS-PAGE procedure utilized a low ionic strength solubilization buffer that resuited in a lower percentage of free chl (12 +-2%). An alternative explanation relates to the concentration dependence of the atrazine response. Lemna may require atrazine concentrations greater than 0.25ppm to affect chlprotein composition, although 0.25ppm was sufficient to alter thylakoid lipid composition (Grenier et al. 1987). Treatment of Lemna with 0.25 ppm atrazine may be analogous to barley treated with 0.03 ppm atrazine (Fig. 3) where chl-protein composition was not affected significantly. The atrazine effects on maximum photosynthetic electron transport measured in vitro were dependent on the developmental stage of the leaf tissue at the time of herbicide application. Sublethal atrazine had no effect on photosynthetic electron transport in primary leaves that were fully developed prior to the herbicide treatment (Fig. 8A, Table 1). In contrast, electron transport was stimulated in the second main shoot leaves that expanded in the presence of 0.07ppm atrazine (Fig. 8B, Table 1). Stimulation of photosynthetic electron transport by PS II herbicides was observed previously when wheat

seedlings were germinated in the presence of BAS 13-38 (Bose et al. 1984). The biochemical basis for this observation remains unclear. Sublethal atrazine stimulated both PS II and PSI activity (Table 1) but did not affect the levels of P-700, cyt f or atrazine binding sites (Table 2), an indication that stimulation of electron transport was not caused by changes in the concentration of the major electron transport complexes. Components not quantitated in these studies, such as plastoquinone and plastocyanin, could be responsible for the stimulation of electron transport in second main stem leaves of atrazine treated plants. Membrane lipids may also be involved because thylakoid membrane lipid composition, particularly the unsaturated fatty acyl lipids, has been correlated with photosynthetic activity (Laskay and Lehoczki 1984). Thylakoid membranes from triazine-resistant (Pillai and St. John 1981, Burke et al. 1982) and triazine-acclimated (Mattoo et al. 1984) plants are known to contain elevated levels of unsaturated fatty acyl substituents. Sublethal effects of PS II herbicides are commonly described as a shade response because of similarities between low irradiance, herbicideacclimated, and herbicide-resistant plants. Increased thylakoid membrane grana stacking and lowered chl a/b ratio are characteristic responses to low irradiance (Leong and Anderson 1984, Davies et al. 1986, De la Torre and Burkey 1990a) and PS II herbicides (Fedtke et al. 1977, Lichtenthaler et al. 1982, Bose et al. 1984, Mattoo et al. 1984). However, other parameters clearly show that irradiance and atrazine treatments have different effects. First, differences exist in the relationship between chl a/b ratio and photosynthetic electron transport. Both chl a/b ratio and chloroplast electron transport activity are reduced under low irradiance conditions (De la Torre and Burkey 1990a,b), but electron transport was either unaffected or stimulated by sublethal levels of atrazine that lowered chl a/b ratio (Bose et al. 1984, Figs. 5 and 8). Similarly, the number of PS II reaction centers and the cyt b6/f complexes in barley thylakoid membranes are sensitive to irradiance (De la Torre and Burkey 1990b), but were unaffected by sublethal atrazine treatments (Table 2). Finally, irradiance and atrazine have different

effects on the distribution of chl between the chl-protein complexes. Irradiance affects PS II antenna size by regulating CPa and LHC-II levels with no effect on the total PSI chl (De la Torre and Burkey 1990a). In contrast, atrazine induces a redistribution of chl between PS II and PSI. The characteristics of triazine-resistant weeds are also quite diverse. Typically, triazineresistant weeds have low chl a/b ratios, elevated LHC-II levels and grana stacking, and lowered electron transport (Burke et al. 1982, Vaughn and Duke 1984, Jursinic and Pearcy 1988), but there are notable examples of resistant weeds with none of these properties (Schonfeld et al. 1987). The plasticity of the thylakoid membrane system is evident from this wide range of responses to different conditions.

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