Metabolic effects in rapeseed (Brassica napus L.) seedlings after root exposure to glyphosate

PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 89 (2007) 220–229 www.elsevier.com/locate/ypest

Metabolic effects in rapeseed (Brassica napus L.) seedlings after root exposure to glyphosate Iben Lykke Petersen a,*, Hans Christian Bruun Hansen a, Helle Weber Ravn b, Jens Christian Sørensen a, Hilmer Sørensen a a

Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Department of Terrestrial Ecology, National Environmental Research Institute, University of Aarhus, Vejlsøvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark Received 6 February 2007; accepted 25 June 2007 Available online 5 July 2007

Abstract Metabolic effects in rapeseed (Brassica napus L.) seedlings after root exposure to sublethal concentrations of glyphosate were examined in order to evaluate the possibilities of using a response pattern in plants as a measure of exposure to glyphosate through the growth media, more sensitive than the well-known biomarker shikimate. Rapeseed seedlings were grown in hydroponic nutrient solutions containing varying sublethal concentrations of glyphosate (1–50 lM). After 9 days of glyphosate exposure, the shoots of the seedlings were analysed with respect to the effects on selected metabolites downstream from the primary affected metabolite shikimate, which accumulated linearly in response to glyphosate exposure (from 0 to 126 lmol/g DW). The selected metabolites analysed, comprising the free amino acids, and the glucosinolates derived therefrom, showed complex patterns in response to glyphosate exposure. Most noteworthy was though that they responded at the lowest concentrations of exposure to glyphosate (1 lM), where no visual effects, decrease in shoot DW or shikimate could be detected, indicating that a biomarker response more sensitive than that of shikimate can be established for plants exposed to glyphosate.  2007 Elsevier Inc. All rights reserved. Keywords: Glyphosate; Root uptake; Shikimate pathway; Aromatic amino acids; Glucosinolates

1. Introduction Glyphosate (N-(phosphonomethyl)glycine) is a foliar applied herbicide that is usually inactivated in the soil by adsorption to soil minerals and by rapid microbial degradation [1]. In situations of glyphosate contamination from point sources, spills or repeatedly use of glyphosate, the result may be an accumulation of the herbicide in the top soil layer, and following sublethal concentrations of glyphosate will be available for the plant roots [2]. This can also be the case when fertilising agricultural soils with phosphate, where glyphosate can be released due to competition for adsorption sites between phosphate and glyphos*

Corresponding author. Fax: +45 35332398. E-mail address: [email protected] (I.L. Petersen).

0048-3575/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.06.009

ate [1]. The commonly used methods to detect soil pollution are soil analyses. These analyses are usually rather costly and in addition they do not express the bioavailability or leachability of glyphosate. Hence, there is a need for development of different bioassays and alternative ways to screen for and/or detect and map soil pollution sceneries. Shikimate is a well-known biomarker for glyphosate exposure [3–5], but in order to develop a bioassay more sensitive than shikimate to detect soils with elevated levels of glyphosate, it is important to know the physiological reactions of the plants to sublethal concentrations of glyphosate taken up via the roots, since the reaction may vary at different sublethal concentrations. Therefore, it is relevant to investigate whether there is a dose–response relationship for selected metabolites other than shikimate.

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The effects from glyphosate is generally considered to be as those from a nonselective herbicide that is rapidly translocated throughout the plant via the phloem. Even though it can take up to several days or weeks for plant death to occur, specific biochemical effects can often be observed within a few hours following glyphosate application [6]. The mode of action of glyphosate comprise inhibition of the transformation of shikimate into chorismate, and thereby glyphosate inhibits the synthesis of shikimate derived aromatic compounds, and finally the result is reduced growth and premature cellular death [7,8]. The enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS, E.C. 2.5.1.19), which catalyzes the formation of 5-enolpyruvylshikimate 3-phosphate (EPSP) from phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P), is assumed to be the primary target of glyphosate in plants [9]. Thereby, it inhibits the shikimate pathway, which produces the important branch point intermediate, chorismate, from erythrose-4-phosphate and PEP. Chorismate is then the biosynthetic precursor for anthranilate (oaminobenzoic acid), prephenate, isoprephenate and various other aromatic compounds (Fig. 1). Prephenate is thus precursor for tyrosine and phenylalanine, anthranilate is precursor for tryptophan and isoprephenate is precursor for the group of m-carboxysubstituted aromatic amino acids and metabolites thereof [10]. The aromatic amino acids and chorismate are also precursors for a great range of other aromatic compounds [10], and it is assumed that up to 60% or more of the ultimate plant mass (dry weight) is represented by molecules once shuffled through the shikimate pathway [11]. Plants belonging to the Brassicaceae (Cruciferae) family accumulate also an important group of compounds that derive from the aromatic amino acids, namely the aromatic glucosinolates. This group of bioactive compounds is characteristic for, and always present in, all plants belonging to this family [12–16]. Hence, glyphosate exposure leads to a cascade of events affecting aromatic amino acids and derived metabolites. With glyphosate inhibition of the EPSPS enzyme, the results are an accumulation of high levels of shikimate [3], and this physiological effect has increased the interest for using shikimate as a (bio)marker for the herbicidal effects of glyphosate [3–5]. Even as it is well-known that shikimate accumulates in response to glyphosate treatment, starting at sublethal concentrations [4,5], informations are insufficient concerning potential effects of glyphosate on the levels of aromatic amino acids and on the allelochemicals as, e.g., glucosinolates derived therefrom. The aromatic amino acids represent a group of metabolites close to the point of glyphosate inhibition, and are already known to decrease in concentration in response to glyphosate exposure [7,8]. Glucosinolates can be considered as a metabolic end-point downstream from shikimate, chorismate and the aromatic amino acids, and therefore it is interesting to investigate if the effect from glyphosate inhibition can be traced as expected. In order to investigate this, a hydroponic system was chosen for this initial study

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as opposed to a true soil system, in order to avoid glyphosate adsorption to the soil, and to ensure glyphosate uptake at known sublethal concentrations. Brassica napus L. (rapeseed) was used as a model plant, since it belongs to the glucosinolate containing plant family Brassicaceae. The objectives of this work were to examine the metabolic effects on rapeseed seedlings after root exposure to glyphosate at sublethal concentrations. In order to evaluate the possibilities of using a response pattern in rapeseed seedlings as a measure of exposure to glyphosate through the growth media, it was desired to determine the levels of the well-known affected metabolite shikimate, as well as specific metabolites derived from the shikimate pathway (Fig. 1). The metabolites investigated were the free amino acids, especially the aromatic amino acids, and glucosinolates as an example of allelochemicals downstream from shikimate and the aromatic amino acids, with focus on phenethyl- and indol-3-ylmethylglucosinolates biosynthetic derived from phenylalanine and tryptophan, respectively. 2. Materials and methods 2.1. Reagents Glyphosate (N-(phosphonomethyl)glycine) 95% purity, and shikimic acid, min. 99% purity, were purchased from Sigma (St. Louis, MO). All other chemicals were of proanalytical grade. Amino acids and glucosinolates were from the laboratory collection and treated by standard procedures [10,14,17,18]. 2.2. Growth and glyphosate treatment of rapeseed Seeds of Brassica napus L. cv. Pollen were purchased from Cetiom: Pilote Pessac (Pessac, France). The seeds were germinated on moist filter paper in a tray covered with plastic film for 11 days in a greenhouse with a 18 C/15 C (16/8 h) day/night temperature regime. In the following steps the seedlings were transferred to plastic pots (5 seedlings per pot) containing 625 mL nutrient solution (0.20 mM KH2PO4, 0.20 mM K2SO4, 0.30 mM MgSO4Æ7H2O, 0.10 mM NaCl, 0.30 mM Mg(NO3)2Æ6H2O, 0.90 mM Ca(NO3)2Æ4H2O, 0.60 mM KNO3, 0.05 mM Fe(III)–EDTA–Na, 7.0 lM MnCl2Æ4H2O, 0.7 lM ZnCl2, 0.8 lM CuSO4Æ5H2O, 2.0 lM H3BO3, 0.8 lM Na2MoO4Æ2H2O [19]). The pots were aerated with filtrated compressed air. After 7 days, the nutrient solution was replaced with new nutrient solution, and the glyphosate treatment was started by adding technical grade of glyphosate to achieve initial concentrations of 0, 1.0, 5.0, 10, 20, 30 and 50 lM (4 replicates (A–D) for each treatment). Nutrient solutions were replaced every 3 days with fresh solutions of the same glyphosate concentrations. After 9 days of glyphosate treatment the plants were removed, separated into root and top, and freeze dried. The biomass was determined as freeze dried tissues.

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I.L. Petersen et al. / Pesticide Biochemistry and Physiology 89 (2007) 220–229 Folic acids O

CH 3

O

H3CO

CH3

H3CO

R

NH N

O HN

O

CH3

NH COO-

CH 3

O

COO-

COO-

HO

Vitamin K

Dihydropteroate

H2 N

CH3

O

R R

N

Ubiquinone

H3 C

alpha-Tocopherole Vitamin E O

O H3C COOCOO -

NH3

OH

p-Hydroxybenzoate

+

H3C

o-Succinylbenzoate

COO-

Plastoquinone

COO -

-

OOC

O CH2

-OOC

C

OH

O

Isoprephenate COO-

COO-

COO-

HO

Homogentisate

OH

CHORISMATE

Prephenate O O

COO-OOC NH3+

OH

O

COO -

OH OH

-OOC

R O

p-Aminobenzoate

COO-

CH3

COOCOO

-

NH3+ NH 3+

OH

m-Carboxy- m-Carboxytyrosine phenylalanine

Anthranilate

NH3+

NH3+

NH3+

COO -

m-Carboxyphenylderivatives

4-Hydroxyphenylpyruvate

Phenylpyruvate

COO-

COO-

NH

Cyanogenic glycosides

OH

Phenylalanine

Tryptophan

Tyrosine OH

Alkaloids Peptides

R

C

S O

NOSO2O-

Proteins Glucosinolates

COO-

Biogenic amines COO-

Nitriles

Various products

Cinnamate

Cinnamoylderivatives Lignins

Ascorbigens

Flavonoids

Antocyanins

Fig. 1. Essential plant metabolites derived from chorismate. The metabolites analysed in this study are highlighted with grey.

2.3. Extraction 2.3.1. Crude extract The initial extraction procedure, with use of internal standards [17], was performed according to described standard procedures [17]. Two hundred and fifty milligram ground freeze dried plant material (replicate A only) was transferred to a centrifuge glass and 100 lL internal standards (7 mM glucotropaeolin, 7 mM epi-glucobarbarin and 113.6 mM trigonellinamide) were added. Extraction (3 · 1 min) was performed in 3 · 3 mL 70% boiling methanol using an Ultra Turrax T 25 (Janke & Kunkel, Staufen,

Germany) to homogenize the plant materials. The homogenates were centrifuged in a table centrifuge at 2000g for 3 min and the supernatants thus obtained were pooled and evaporated to dryness by filtrated compressed air. The residues were then redissolved in 5.0 mL Milli-Q water (Purification Pak, Millipore, Molsheim, France) resulting in the solution referred to as the crude extract. 2.3.2. PSE crude extract In order to automate the extraction procedure, crude extracts of replicates A-D was made using a Pressurised Solvent Extractor (PSE), ASE200, from Dionex (Sunny-

I.L. Petersen et al. / Pesticide Biochemistry and Physiology 89 (2007) 220–229

vale, CA). Two hundred and fifty milligram ground freeze dried plant material (replicate A–D) was transferred to a 1 mL extraction cell and 100 lL internal standards (7 mM glucotropaeolin, 7 mM epi-glucobarbarin and 113.6 mM trigonellinamide) were added. Extraction was performed using 100% methanol according to the following conditions: Preheat: 5 min; heat: 5 min; static: 5 min; flush%: 30 vol.; purge: 100 s; cycles: 3; pressure: 1500 psi; temperature: 90 C. The extracts obtained were evaporated to dryness by filtrated compressed air, and the residues were then redissolved in 4.0 mL Milli-Q water. This is referred to as the PSE crude extract. 2.4. Separation of plant compounds 2.4.1. Amino acids Part of the crude extract (3.0 mL) was in the following step subjected to ion exchange chromatography according to the procedures described by Sørensen et al. [17], in order to isolate the neutral and acidic amino acids. Aqueous suspensions (1 mL; 1:1) of (A) CM Sephadex C-25 (H+) and (B) Dowex 50 W · 8, 200–400 mesh (H+) were packed into 1 mL plastic columns supplied with filter disks of silica material at the bottom. The columns were regenerated by use of 10 mL of 2 M acetic acid (A) and 15 mL of 1 M HCl (B), respectively, followed by wash with water until neutral pH according to standard procedures [17]. Column A was placed at the top of column B and the collected columns were placed in a vacuum manifold (Supelco, Bellefonte, PA). After application of the crude extract (3.0 mL), the columns were washed with 3 · 4 mL Milli-Q water. Finally, the B column was eluted separately with 3 · 5 mL 1 M pyridine according to standard procedures [17], to release the retained amphoions including neutral and acidic amino acids [14,18]. The eluates obtained were evaporated to dryness by filtrated compressed air, and the residues were then redissolved in Milli-Q water. An internal standard (30 lL 4.60 mM N-valine) was added to the redissolved B eluate, and the amino acids was then transformed into dinitrobenzene derivatives by the Sanger method prior to analysis by micellar electrokinetic capillary chromatography (MECC) [17]. 2.4.2. Glucosinolates The glucosinolates were isolated by standard procedures from the crude extract [17], where the glucosinolates oncolumn were transformed into desulfoglucosinolates prior to described MECC procedures [14–17,20–22].

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yses, the capillary was flushed after each analysis with 1 M NaOH for 2 min, Milli-Q water for 1 min and separation buffer for 5 min. 2.5.1. Analysis of PSE crude extract The PSE crude extract was concentrated 5 fold before analysis (500 lL evaporated to dryness by filtrated compressed air and redissolved in 100 lL Milli-Q water), and the analyses were performed according to standard procedures [14–17]. The capillary was a 645 mm · 0.05 mm i.d. barefused silica capillary with a UV detection window placed on-column at a position of 560 mm from the injection end. The signal wavelength was set at 230 nm. Samples were introduced from the cathodic end of the capillary by vacuum injection for 1 s at 40 mbar. The separation buffer (35 mM cholate, 50 mM taurine, 100 mM Na2HPO4Æ2H2O, 2% 1-propanol, pH 7.3) was filtered through a 0.20 lm membrane filter before use. The analyses were performed at 18 kV and 30 C for 40 min. 2.5.2. Analysis of shikimate Shikimate was detected in the HPCE analysis of the PSE crude extract as described elsewhere [3]. In short, this analysis enables for the detection of shikimate within 15 min, and with a cLOD of 24.4 lM, and a recovery for the extraction procedure of 99.8% [3]. 2.5.3. Analysis of amino acids The capillary was a 960 mm · 0.075 mm i.d. barefused silica capillary with a UV detection window placed on-column at a position of 840 mm from the injection end. The signal wavelength was set at 360 nm. Samples were introduced from the cathodic end of the capillary by vacuum injection for 2 s at 50 mbar. The separation buffer (60 mM TTAB, 20 mM Na2HPO4*2H2O, 18 mM Na2B4O7, 13% 1-propanol, pH 9.6) was filtered through a 0.20 lm membrane filter before use. The analyses were performed at 18 kV and 50 C for 55 min. 2.5.4. Analysis of glucosinolates The capillary used for the MECC analyses of desulfoglucosinolates [20,22] was a 645 mm · 0.05 mm i.d. barefused silica capillary with a UV detection window placed on-column at a position of 560 mm from the injection end. The signal wavelength was set at 230 nm. Samples were introduced from the anodic end of the capillary by vacuum injection for 1 s at 50 mbar. The separation buffer (250 mM cholate, 200 mM borate, pH 8.5) was filtered through a 0.20 lm membrane filter before use. The analyses were performed at 12 kV and 60 C for 40 min.

2.5. High performance capillary electrophoresis (HPCE) analyses

2.6. Statistics

All HPCE analyses were performed using a HewlettPackard HP3D CE capillary electrophoresis system (model G1600AX, Hewlett-Packard, Waldbronn, Germany) equipped with a diode-array detector (DAD). For all anal-

Biomass of shoots and roots, as well as concentration of shikimate in shoots were calculated on the basis of four replicates (n = 4), and standard deviations were calculated. Screening for changes in free amino acids and glucosino-

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lates were done on the basis of one replicate (n = 1). In order to ensure correct determinations of concentrations, internal standards were added at the beginning of the extraction. Concerning the amino acids, norvaline was used as internal standard. Concerning the glucosinolates, two internal standards were added and used as a point of reference when calculating the concentrations. Moreover, response factors [17] for each glucosinolate were used for the calculations.

The roots show the highest sensitivity to glyphosate, with an approximate reduction of 83% of dry weight (DW), as opposed to 43% DW reduction in the shoots (Fig. 3). These results are supported by earlier results showing similar differences in root and shoot sensitivity [23–25]. Both visual and DW results show a tendency towards improved growth (hormesis) for the plants exposed to the lowest glyphosate concentrations. Similar trends have been reported by Pline et al. [25] and Thomas et al. [26].

3. Results and discussion

3.2. Metabolites in the crude extract

3.1. Growth response to glyphosate treatment

The HPCE analyses of the crude extracts revealed differences in the groups of compounds that increased/decreased in response to glyphosate treatment (Fig. 4). Most pronounced was the accumulation of shikimate (see below), which increased to a level where it was the most dominating metabolite detected in the electropherogram of the crude extract from plants exposed to the highest glyphosate concentrations. Three other groups of compounds could be seen in the crude extract, consisting of the glucosinolates as well as the sinapoylderivatives [17,27,28] and aromatic compounds including a group of flavonoids migrating prior to the glucosinolates (Fig. 4). The changes within the group of glucosinolates have been investigated to further detail as described below. The group of flavonoids [17,28] and sinapoylderivatives [17,27] showed minor changes, even though these groups both derive from chorismate (Fig. 1) and, therefore, are expected to be affected by glyphosate. These groups are not further analysed within this study. Range finding experiments, including screening

After 9 days of glyphosate treatment, the rapeseed seedlings were harvested. At this time it was visually possible to detect the glyphosate affected plants from treatment with 20 lM glyphosate and higher tested concentrations by comparison with a control plant, though it should be mentioned that considerable visual variations within replicates of the same treatment occurred for the plants treated with 20 lM glyphosate and higher. Plants exposed to glyphosate treatments of 1, 5 and 10 lM glyphosate could visually not be differentiated from the control plants at the day of harvest (Fig. 2). Preliminary experiments with exposures to glyphosate concentrations higher than 50 lM resulted in a total knock out of the plants; whereas the plants exposed to the highest concentration of glyphosate in this experiment (50 lM) resulted in a significant decrease in biomass production as well as obvious necrotic leaves, especially the cotyledons.

Fig. 2. Photograph of rapeseed (B. napus L.) seedlings grown in hydroponic solutions with varying glyphosate concentrations as indicated, 9 days after treatment start. (A–D) denotes the four replicates (=pots) made for each treatment.

I.L. Petersen et al. / Pesticide Biochemistry and Physiology 89 (2007) 220–229 1.8 Shoots 1.6

1.4

1.2

Dry weight (DW) (g)

1.0

0.8

0.6 Roots

0.30 0.25 0.20 0.15 0.10 0.05 0.00 0

10

20

30

40

50

Glyphosate concentration in nutrient solution (µM) Fig. 3. Growth response of rapeseed (B. napus L.) seedlings to glyphosate concentration in the hydroponic nutrient solution 9 days after treatment start. Each data point represents the mean dry weight of plants from four pots (A–D). Error bars represents standard deviation (n = 4).

of ubiquitous compounds and allelochemicals [29], had also revealed a whole range of effects on metabolites, including amino acids, phenolic compounds, shikimate and S-containing compounds, to which the glucosinolates belong. On the basis of these preliminary results, together with HPCE analyses of the crude extract, it was chosen to investigate the responses to sublethal concentrations of glyphosate treatment among the metabolites downstream from chorismate, namely the free amino acids as well as the glucosinolates. Shikimate was analysed as a point of reference. 3.3. Shikimate accumulation in response to glyphosate treatment Shikimate, an intermediate of the shikimate pathway, accumulates according to the literature in response to glyphosate treatment [4,5,8,25,26,30–43]. In the present experiments, shikimate analysed by the MECC procedure [3] was not detectable in the shoots of the control plants as well as in the plants exposed to 1 lM glyphosate. In the range of 10–50 lM glyphosate exposure, shikimate accumulation showed a linear response (R2 = 0.985) to the glyphosate dose applied to the roots (Fig. 5). The results confirm that glyphosate has been taken up by the plants via the roots, even at sublethal concentrations where there was no visual sign of effect on the plants, or decrease in shoot DW. 3.4. Analysis of amino acids The amino acids are analysed as free amino acids, which is the non-protein bound amino acids. The general trend in the content of amino acids seen in relation to the glyphosate treatment was that higher glyphosate treatment resulted in increasing concentrations of amino acids (Fig. 6). In this

Shikimic acid Glucosinolates

50

225

Sinapoylderivatives

160

Absorbance (mAU)

40

30 μM 30

20 μM 10 μM

20 5 μM 1 μM

10

0 μM 0 5

10

15 Time (min.)

20

Shikimatein shoots (μmol/g DW)

50 μM

140 120 100 80 60 40 20 0

25

0

10

20

30

40

50

Glyphosate concentration in nutrient solution (μM) Fig. 4. Electropherograms showing HPCE analyses of the crude extracts at 230 nm. The first group of compounds highlighted (from the left) consists of glucosinolates. Following, a peak representing shikimate is absent/increasing, and last, a group of compounds consisting of sinapoylderivatives is highlighted.

Fig. 5. Shikimate levels in shoots of rapeseed (B. napus L.) seedlings in response to glyphosate concentration in the hydroponic nutrient solution 9 days after treatment start. Each data point represents the mean concentration of shikimate from shoots of plants from four pots (A–D).

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I.L. Petersen et al. / Pesticide Biochemistry and Physiology 89 (2007) 220–229 60

5 Phe Trp Tyr

50

3

40

2

30 Gln GABA/Pro Glu

1

20

10

0

0 30 30 20

Ala Val Ile Leu

Asn Asp Thr

20

Aminoacid in shoots (μmol/g DW)

Amino acid in shoots (μmol/g DW)

4

10 10

0

0

0

10

20

30

40

50

0

10

20

30

40

50

Glyphosate concentration in nutrient solution (μM)

Fig. 6. Amino acids in shoots of rapeseed (B. napus L.) seedlings in response to glyphosate concentration in the hydroponic nutrient solution 9 days after treatment start. The amino acids are grouped according to their biosynthetic family of origin: (Top left) Aromatic amino acids; (bottom left) aspartate family; (top right) glutamate family; (bottom right) ‘‘pyruvate’’ family.

respect, the amount of aspartic acid (Asp), gamma-aminobutyric acid (GABA)/proline (Pro), glutamine (Gln) and asparagine (Asn) increased more than 5–10-fold, when comparing the plants exposed to the highest glyphosate concentration (50 lM glyphosate) with the control plants. Looking only at the aromatic amino acids, the results reveal no clear pattern in the contents seen in relation to exposure to glyphosate, but the concentration of all amino acids appeared to be affected even at the lowest glyphosate concentration applied (1 lM). Most pronounced is the increase in phenylalanine concentration in response to glyphosate concentration of 1 and 10 lM. Glyphosate blocks the biosynthesis of the aromatic amino acids (Fig. 1), but the results reveal no clear decrease in the content of the aromatic amino acids phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr), when their concentrations in plants exposed to glyphosate are compared to the concentrations in the control plants. Phenylalanine shows a very clear response to glyphosate exposures of 1 and 10 lM in the nutrient solution, but then the concentration seems to decrease and at 50 lM glyphosate exposure, the concentration of phenylalanine is below the concentration in the control plant. There seems to be a similar trend in the development of phenylalanine and tyrosine, where the increase in the concentration at the lowest exposures to gly-

phosate (1 and 10 lM) is followed by a decrease in the concentration at exposures to higher glyphosate concentrations (20–50 lM). The decreasing trend is in agreement with the theory on the mode of action of glyphosate, but the increase is not. The trend for tryptophan seems to be increasing rather than, as expected, decreasing. A feasible explanation could be, as stated by Amrhein et al. [8], that tryptophan is the least sensitive of the aromatic amino acid to glyphosate inhibition, partly due to differences in enzyme affinities for chorismate. Other workers have also reported a greater effect of glyphosate on phenylalanine and tyrosine, rather than on tryptophan. Forlani et al. [44] analysed the amino acid pools in cells of Nicotiana plumbaginifolia four days after the addition of 100 lM glyphosate to the culture medium. Thereby they found a reduction in the concentration of tyrosine and phenylalanine corresponding to 59% and 77%, respectively, whereas tryptophan was reduced by only 13% [44]. Wang [37] investigated the effect of glyphosate on aromatic amino acids in the shoots of Cyperus rotundus, and found that the tyrosine and phenylalanine concentration three days after treatment did not differ significantly from the control, whereas tryptophan concentration was significantly higher in glyphosate treated shoots compared to control shoots. These findings, together with the results

of this present work, support the theory concerning differences in enzyme affinities for chorismate [8]. The increasing trend for especially phenylalanine at very low glyphosate exposures is supported by the results from Ravn et al. [29] who also found an increasing trend for phenylalanine in Apera spica-venti in response to a foliar applied glyphosate treatment. Even though glyphosate should not have any primary effect on the amino acids of the non-aromatic biosynthetic families, a general increase in concentration is seen for some of these compounds when the plants are exposed to increasing concentrations of glyphosate, in particular Asp, GABA/Pro and Gln (Fig. 6). One explanation for this could be a combined effect on metabolic regulations and an increased protein hydrolysis in response to the glyphosate inhibition of the shikimate pathway and its metabolites [37]. Another explanation could simply be, as proposed by Jaworski [7], that a specific block in the aromatic amino acid biosynthesis results in a slow down in protein synthesis, and therefore the demand for other amino acids decreases, causing the pool of free amino acids to increase. Ravn et al. [29] found similar increasing trends in the amounts of the free aliphatic amino acids Glu, Gln, Pro, Val, Leu/Ile, Thr and Lys in Apera spica-venti in response to a foliar applied glyphosate treatment. 3.5. Analysis of glucosinolates Considering the group of amino acid derived allelochemicals, the glucosinolates are of special interest for Brassicaceae. In theory, the synthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan should be reduced as a function of increased glyphosate concentration, and following this, the formation of phenetyl- and indol-3-ylmethylglucosinolates should also be reduced, since the precursors for these compounds are phenylalanine and tryptophan, respectively (Fig. 1). As discussed above, other factors may play a role though, such as protein hydrolysis or slow down in protein synthesis. The main glucosinolates seen in B. napus (rapeseed) plants used in this experiment comprises the aliphatic glucosinolates (glucobrassicanapin and progoitrin) derived from methionine, phenetylglucosinolate (gluconasturtiin) derived from phenylalanine, and indol-3-ylmethylglucosinolates (glucobrassicin, neoglucobrassicin, and 4-methoxyglucobrassicin) derived from tryptophan [13–16] (Fig. 7). It is expected that the amount of gluconasturtiin, glucobrassicin, neoglucobrassicin and 4-methoxyglucobrassicin should decrease following a decrease of their precursors phenylalanine and tryptophan due to glyphosate treatment of the plants. Gluconasturtiin was quite stable in concentration, not decreasing significantly in concentration as expected. Likewise for the indol-3-ylmethylglucosinolates, glucobrassicin, neoglucobrassicin and 4-mehtoxyglucobrassicin, no clear signs of effect were to be detected. The trend for neoglucobrassicin and 4-methoxyglucobrassicin seemed though to be decreasing at the lowest concentrations of exposures, and

Glucosinolates in shoots (μmol/g DW)

I.L. Petersen et al. / Pesticide Biochemistry and Physiology 89 (2007) 220–229

6

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Glucobrassicin * Neoglucobrassicin * 4-Methoxyglucobrassicin * Gluconasturtiin ** Glucobrassicanapin *** Progoitrin ***

4

2

0 0 1 5 20 30 50 Glyphosate concentration in nutrient solution (μM)

Fig. 7. Glucosinolates in shoots of rapeseed (B. napus L.) seedlings in response to glyphosate concentration in the hydroponic nutrient solution 9 days after treatment start. The glucosinolates are marked according to their biosynthetic family of origin, where * comprises the glucosinolates derived from Trp, ** comprises the glucosinolates derived from Phe, and *** comprises the glucosinolates derived from Met.

then increasing at the highest concentrations of exposures to glyphosate, which indicates a sensitive, but not linear, response to glyphosate exposure, as also seen for especially phenylalanine. The concentrations of the aliphatic glucosinolates vary with glyphosate exposure, but the overall picture is neither increasing nor decreasing as would also be expected (Fig. 7). The expected clear effects on the phenetyl- and indol-3-ylmethylglucosinolates due to exposure to glyphosate can not be documented by results obtained in the present investigations, and there is no simple dose–response pattern, but as stated above, there are indications for a sensitive reaction to low glyphosate concentrations. This reaction seems though to be less sensitive than that for phenylalanine. To our knowledge, this is the first investigation to trace the effects of glyphosate on B. napus glucosinolates. A summary of the presented results can be seen in Table 1, showing a synthesis of the screening results, where the changes in concentrations have been shown as percentage of the concentrations in the control plants. The results for the free amino acids and glucosinolates have been grouped according to their biosynthetic origin. Considering the figures for the biomass these indicate an increase in the biosynthetic processes up to 10 lM glyphosate in the nutrient solution, while at glyphosate concentrations higher than 10 lM catabolic processes dominate with reduced weight/size of the plants as the results (Fig. 2). These glyphosate concentrations give also a very pronounced increase in the shikimate concentrations in the shoots. Most noteworthy is though the trends seen for the groups of amino acids and glucosinolates derived from chorismate (aromatic amino acids, indol-3-ylmethylglucosinolates (derived from Trp) and phenethylglucosinolate (derived from Phe)), where it becomes very clear that even at the

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Table 1 Overview and synthesis of screening results presented in Figs. 3 and 5–7 Glyphosate concentration in nutrient solution (lM)

General

Free amino acids

Glucosinolates

Biomass (shoots)

Shikimate

Aromatic

Aspartate family

Glutamate family

‘‘Pyruvate’’ family

Derived from Trp

Derived from Phe

Derived from Met

0 1 5 10 20 30 50

100 102 104 106 85 87 57

nd nd 100 114 977 2085 5083

100 172 — 233 129 114 85

100 86 — 103 132 163 233

100 87 — 100 153 198 321

100 95 — 120 125 132 160

100 72 75 — 60 87 75

100 54 73 — 72 84 61

100 53 150 — 434 336 239

Changes in weight of shoots/concentration of metabolites in shoots expressed as percentage of control; nd, not detectable.

lowest concentration of glyphosate exposure to the roots (1 lM), major metabolic changes occur. At the lowest concentrations of glyphosate exposure, the concentration of aromatic amino acids seems to increase dramatically, whereas the glucosinolates derived therefrom seem to decrease in concentration. At the highest concentration of glyphosate exposure, the pattern is clear and as expected, where the metabolites derived from shikimate and chorismate decrease in concentration whereas the other metabolites increase in concentration. 4. Conclusions This study demonstrates that the trend in metabolic changes can differ significantly from plants exposed to very low concentrations of glyphosate compared to plants exposed to higher, but still sublethal, concentrations of glyphosate. The present study confirms the potential of shikimate as a biomarker for the exposure of plants to glyphosate both taken up via roots and/or leaves, and the results show that shikimate has a linear response to the glyphosate dose applied to the roots. Finally, it is demonstrated that glyphosate even at the lowest sublethal concentration (1 lM) greatly affects the composition and concentrations of the metabolites in the shoots derived from the shikimate pathway, even when no visual effects can be seen, or shikimate can be detected. Since these responses have not shown a linear dose–response pattern, it is not possible to use one single metabolite as a biomarker of glyphosate exposure. In order to achieve a more sensitive response than shikimate can offer, it is necessary to utilise a combination of a range of metabolites sensitive to glyphosate exposure, and for this purpose, post-analytical methods like multivariate data analysis could turn out to be a sensible choice of tool, but this is out of scope for this paper. Acknowledgments The authors gratefully acknowledge the financial support from the Information Centre on Contaminated Sites, Copenhagen, from the Danish Research Council, Grant

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