Proc Natl Acad Sci U S A 110(15): 5812-5817

PMC: PMC3625300

PMID: 23530204

Key role for a glutathione transferase in multiple-herbicide resistance in grass weeds

Lesley Edwards+5 authors

AmGSTF1 and Orthologs Are Highly Expressed in MHR Black-Grass and Annual Rye-Grass.

To determine how MHR affected protein expression, crude extracts from Peldon and WTS black-grass were fractionated on phenyl Sepharose to remove photosynthetic components and the polypeptides present visualized by staining following two-dimensional gel electrophoresis. Protein profiles were essentially identical, except for seven polypeptides with molecular masses around 28 kDa, which were enhanced in the MHR Peldon plants (Fig. 1A). The 28-kDa polypeptides were all identified as isoforms of AmGSTF1 on the basis of their identical peptide fingerprints and the presence of a diagnostic 1,038-Da fragment that yielded the common sequence VFGPAMSTNV following tandem MS (15). Analysis of leaf extracts showed that AmGSTF1 corresponded to 0.2% of the total protein in the MHR plants, being over 20 times more abundant than in the respective WTS weeds. These differences in expression levels were confirmed by Western blotting using an anti-GST serum (Fig. 1B) and by quantitative PCR (qPCR), with transcripts encoding AmGSTF1 isoenzymes being 13 times more abundant in MHR Peldon compared with WTS plants (Fig. 1C). To determine whether orthologs of AmGSTF1 were present in annual rye-grass, the MHR biotype SLR 31, which shows enhanced expression of herbicide-detoxifying CYP enzymes (17), was analyzed. Using conserved GSTF1-like sequences found in black-grass and cereals, eight closely related (83–91% amino acid identity) PCR amplification products were generated. Based on qPCR, these products were 4 times more abundant in the MHR SLR 31 plants than in the corresponding WTS weeds (Fig. 1C). As determined by Western blotting, GSTF1-like polypeptides were also more abundant in extracts from SLR 31 plants and the independent MHR biotype VLR 69 (18), compared with WTS plants (Fig.1B and Fig. S1A). A cDNA encoding a L. rigidum (Lr) LrGSTF1 with 91% identity to AmGSTF1 was then isolated from annual rye-grass by PCR (Fig. 2) and then cloned and expressed in Escherichia coli alongside AmGSTF1. Both recombinant enzymes had similar enzyme activities (Table 1), being highly active GPOXs and showing a limited ability to catalyze the glutathione conjugation of the model GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Consistent with these findings, GPOX activities determined in the MHR annual rye-grass populations were two- to fourfold higher than those determined in WTS plants (Fig. S1B).

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Fig. 1.

Analysis of the GSTs in MHR grass weeds and transgenic Arabidopsis. (A) Analysis of soluble hydrophobic protein extracts from WTS and MHR Peldon black-grass biotypes by 2D gel electrophoresis. The red arrows refer to polypeptides identified by proteomics as AmGSTF1 subunits. (B) Western blots showing immunodetectable GSTF1 polypeptides in MHR black-grass (Peldon) and annual rye-grass (SLR31) and Arabidopsis plants expressing AmGSTF1 (lines 8 and 12) relative to WTS weeds and vector only (V) controls. (C) GSTF1 transcript abundance in WTS and MHR biotypes of black-grass and annual rye-grass as determined by qPCR.

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Fig. 2.

Aligned sequences of GSTF1 orthologs from black-grass (UniProt accession no. {"type":"entrez-protein","attrs":{"text":"Q9ZS17","term_id":"75338940","term_text":"Q9ZS17"}}Q9ZS17) and annual rye-grass. Residues within boxes represent hypothetical active-site residues inferred from the known maize GSTF1 crystal structure (Protein Data Bank accession no. 1axd). Cys120 residue is shaded in gray. Asterisks (*) denote identical amino acid residues. Nonidentical amino acids with similar properties are denoted by : and . .

Table 1.

Activities of black-grass and annual rye-grass GSTF1 enzymes

	Mean enzyme-specific activity, nmol⋅s^⋅mg^protein
Substrate	AmGSTF1	LrGSTF1
CDNB	22.7 ± 0.42	26.3 ± 0.42
Cu-OOH	21.9 ± 0.42	43.5 ± 0.71
Lin-OOH	98.6 ± 15.9	272.6 ± 22.5

CDNB: 1-chloro-2,4-dinitrobenzene. Cu-OOH: cumene hydroperoxide. Lin-OOH: linoleic acid hydroperoxide. Measurements were performed in technical triplicate. Mean enzyme-specific activities are shown ± SD, n = 3.

Expression of AmGSTF1 in Arabidopsis Results in an MHR Phenotype.

The high levels of expression of GSTF1 orthologs in two species of MHR weeds suggested an important role for these proteins in herbicide resistance. To investigate the function of black-grass AmGSTF1, the enzyme was expressed in Arabidopsis, using the constitutive 35S promoter. The plants were similarly transformed with AmGSTL1, a lambda class member of the GST superfamily that is also constitutively enhanced in MHR black-grass (14). The expression of the black-grass GSTs in the homozygote immediate (T1) progeny was confirmed by Western blotting. With AmGSTL1, low levels of a 27-kDa polypeptide were identified in the transgenics that was absent in the controls (Fig. S2A), with the highest-expressing plants (line 16) used for further characterization. The anti-GSTF serum identified 26-kDa polypeptides in control plants (Fig. 1B), corresponding to the expression of known endogenous GSTFs in Arabidopsis (19). In the AmGSTF1 transgenics, additional 28-kDa immunoreactive polypeptides were also determined (Fig. 1B). Two lines showing intermediate (line 8) and high (line 12) expression of the transgene were selected for further analysis. All AmGST transformants, together with the respective controls, were tested for herbicide tolerance, using a combination of spraying whole plants, as well as germination phytotoxicity studies on agar (Fig. 3). The herbicides selected were the chloroacetanilide alachlor that inhibits fatty acid elongation and hence cell division and the PS II inhibitors atrazine (chloro-s-triazine) and chlorotoluron (phenylurea). Other classes of graminicidal herbicides used to control black-grass could not be tested in Arabidopsis, as they were either too toxic (e.g., the sulfonylurea thifensulfuron-ethyl and the diphenyl ether fluoroglycofen-ethyl) or inactive, due to inherent differences in herbicide target sensitivities in monocots and dicots (e.g., the aryloxyphenoxypropionate fenoxaprop-p-ethyl). AmGSTL1 transformants were as susceptible to herbicides as the vector-only controls (Fig. S2B). In contrast, the AmGSTF1 transformants were considerably more resistant to all three herbicides, both in spray and in germination trials (Fig. 3). Importantly, although the transgenic expression of a GST could enhance tolerance to alachlor and atrazine, which both undergo S-glutathionylation as primary steps in their metabolism (13), this is not the case with chlorotoluron, which is detoxified in plants by the combined action of CYPs and UGTs (20). To determine whether the detoxifying glutathione-conjugating activity of AmGSTF1 could contribute to the increased tolerance toward alachlor and atrazine, plant extracts were assayed for GST-conjugating activities. Consistent with the activity profile of the enzyme, both AmGSTF1 transgenic lines showed a 3- to 4.5-fold enhancement in GPOX activity and increased glutathione conjugation of CDNB and the herbicide alachlor (Table 2). The AmGSTF1 expressors also showed increased conjugating activity toward the herbicide atrazine (Table 2). Because AmGSTF1 had no detectable activity with atrazine as a substrate (15), these increases in GST activity had to result from the increased expression of endogenous Arabidopsis enzymes. Similarly, AmGSTF1 expression was also associated with an enhancement in unrelated glycosylating activities in Arabidopsis toward the xenobiotic 2,4,5-trichlorophenol (Table 2). Thus, although the exact route of atrazine detoxification in the AmGSTF1 transformants was not determined, it was demonstrated that two independent routes of bioconjugation known to be involved in the metabolism of this herbicide were both enhanced (13). Recent studies have shown that MHR in grasses is also associated with changes in endogenous antioxidant and secondary metabolism, notably an accumulation of cytoprotectants such as glutathione, flavonoids, and anthocyanins (14). When the transgenic Arabidopsis plants were analyzed using liquid chromatography coupled to MS detection (LC-MS), a range of UV-absorbing metabolites were determined (Fig. 4), which accumulated at higher levels in the AmGSTF1 expressors compared with vector-only controls (Table 2). On the basis of their UV and MS spectra, these compounds were identified as conjugates of the flavonol kaempferol and the anthocyanin cyanidin, respectively (Fig. 4). Levels of glutathione (GSH) were also shown to be modestly enhanced in the AmGSTF1 expressors, although the ratio of reduced to oxidized forms was unaffected relative to controls (Table 2). To determine whether these biochemical changes were associated with a perturbation in gene expression, the transcriptome of the line 12 transgenics was compared with that of wild-type plants, using the Affymetrix GeneChip platform. Ranking the top 50 up- and down-regulated genes showed only minor changes in the transcriptome (Dataset S1). When the top 12 most perturbed genes were used as qPCR biomarkers in the two independent AmGSTF1-expressing plant lines (lines 8 and 12), no consistent changes in gene expression were determined (Fig. S3). This inferred that the changes in biochemistry determined following transformation with AmGSTF1 were not regulated at the level of transcription.

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Fig. 3.

Herbicide resistance of transgenic Arabidopsis expressing AmGSTF1. (A) AmGSTF1 expressors and vector-only controls were germinated on agar containing 2 µM chlorotoluron, alachlor, atrazine, or acetone and maintained for 30 d. (B) AmGSTF1-expressing and vector-only control plants were sprayed with chlorotoluron, alachlor, atrazine, or formulation only at rates of 30 g ai per hectare, 1200 g ai per hectare, and 30 g ai per hectare, respectively, and assessed 9 d after herbicide application.

Table 2.

Biochemical phenotype of two independent lines of Arabidopsis transformed with AmGSTF1 compared with plants transformed with the respective empty vector

	Vector only	Line 8	Line 12
Enzyme activity, nkat⋅mg⁻¹
GST	0.26 ± 0.01	1.01 ± 0.01	1.66 ± 0.02
GPOX	0.02 ± 0.00	0.06 ± 0.00	0.09 ± 0.00
Glutathione reductase	0.434 ± 0.00	0.456 ± 0.01	0.406 ± 0.00
Thiol transferase	0.029 ± 0.01	0.088 ± 0.01	0.097 ± 0.01
Catalase	1,949 ± 33	1,725 ± 163	1,718 ± 264
Antioxidant content, nmol⋅g^FW
GSH	202 ± 5	252 ± 8	299 ± 6
GSSG	9 ± 1	10 ± 1	12 ± 2
Flavonol	564 ± 32	1,086 ± 59	1,237 ± 67
Anthocyanin
Peak 2	115 ± 9	454 ± 32	466 ± 47
Peak 3	122 ± 5	432 ± 9	483 ± 23
Herbicide-conjugating activity, pkat⋅mg⁻¹
Alachlor	3.5 ± 0	7.1 ± 0.2	10.7 ± 0.2
Atrazine	0.015 ± 0.00	0.061 ± 0.01	0.056 ± 0.00
Glucosyl transferase activity, fkat⋅mg⁻¹
2,4,5-Trichlorophenol	15.4 ± 3.1	22.9 ± 1.3	21.1 ± 2.0
Quercetin	16.7 ± 0.8	19.9 ± 2.2	20.9 ± 0.7

Measurements are shown ± SEM, n = 3. FW: fresh weight.

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Fig. 4.

(A and B) Polyphenol content of Arabidopsis plants transformed with (A) vector only or (B) AmGSTF1. Flavonols and anthocyanins were identified by HPLC-MS with reference to earlier published work. Peaks 1 and 4 were rhamnosylated conjugates of the flavonol kaempferol, whereas peaks 2 and 3 were derivatives of the anthocyanin cyanidin.

MHR in Weeds Is Chemically Reversible.

The results of the transgenesis studies showed that AmGSTF1 played a causative role in MHR. It was therefore of interest to identify potential chemical intervention strategies that could disrupt the function of the GST and help restore herbicide sensitivity. Drawing on parallels with MDR in humans, the inhibition of drug-detoxifying GSTs has been a productive target for medicinal chemistry programs (21). Such chemical interventions have also been shown to disrupt GSTs functioning in signaling roles, for example in modulating the c-Jun-N-terminal kinase (JNK) and apoptosis signal-regulating kinase (ASK1) signaling pathways (16, 22). As such, there is good precedence for using inhibitors to disrupt GSTs eliciting resistance through multiple mechanisms. These inhibitors can be subdivided into GSH conjugates and related peptidomimetics that bind in the related glutathione (G) binding site of GSTs and compounds acting on the large hydrophobic (H) binding domain (22). The latter simpler H-site inhibitors developed for cancer chemotherapy (23) were tested for their ability to inhibit GSTF1 activity and to augment herbicide efficacy. These included the classic GST inhibitor ethacrynic acid, as well as compounds based on bromoenol lactone and benzoxadiazole chemistries (Fig. 5A). Each compound was tested for its ability to inhibit the conjugating activity of AmGSTF1 toward CDNB. In parallel, each compound was sprayed onto WTS and MHR Peldon black-grass 48 h before an application of herbicide. By combining the in vitro and in planta screens, several compounds that inhibited GST activities could be discounted from further exploration due to their innate phytotoxicity (e.g., compounds 2 and 3) or a lack of an observable potentiation in herbicide activity (compound 4, Fig. S4). The compound 4-chloro-7-nitrobenzoxadiazole [1, nitrobenzoxadiazole (NBD)-Cl], derivatives of which target GSTPs in tumor cell lines (16, 24), was shown to both inhibit AmGSTF1 (Fig. 5B) and enhance the phytotoxicity of chlorotoluron when presprayed on MHR Peldon black-grass (Fig. 6 A and B). Similarly, NBD-Cl enhanced the herbicidal activity of the ACCase-inhibiting graminicides fenoxaprop-p-ethyl and clodinafop-propargyl when applied to Peldon plants, with similar results obtained with the independent MHR Spain biotype (Fig. 6 C–F). Consistent with results obtained with this class of chemistry in MDR cells (16, 24), NBD-Cl exerted its effects without causing overt secondary toxicity. Thus, both black-grass (Fig. 6 A and B) and wheat (Table S1), showed no leaf damage or growth inhibition when exposed to NBD-Cl. Using simple nucleophilic aromatic substitution chemistry, a series of NBD derivatives were prepared, bearing a variety of leaving and activating groups, and used in spray trials (Fig. 7). On the basis of the results of this limited screen (Fig. S5), a nitro group proved to be the most viable activating group. Importantly for future agrochemical optimization, the enhancement of herbicide activity showed a dependence on chemical structure, notably on the nature of the leaving groups (alkoxy and thiol > amino). Intriguingly, modifying the NBD pharmacophore with similar modifications to those shown to potentiate activity in MDR proved ineffective in counteracting MHR. For example, the active GSTP1 inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)-hexanol (NBDHEX) (compound 5, Fig. S5A) (16) proved ineffective when used with chlorotoluron against MHR black-grass, possibly due to its inability to be taken up by intact leaves.

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Fig. 5.

Selected GST inhibitors tested in this study. (A) Inhibitor chemical structures. (B) Their efficacy against black-grass and annual rye-grass GSTF1s as determined from IC₅₀ values. 1, NBD-Cl; 2, ethacrynic acid; 3, cyanuric chloride; 4, bromoenol lactone.

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Fig. 6.

Effect of NBD-Cl on herbicide resistance in black-grass. (A and B) For studies with chlorotoluron, (A) WTS and (B) MHR Peldon black-grass plants were treated at 12 d with either formulation or NBD-Cl (270 g ai per hectare), before an application of either formulation only (Form) or 500 g ai per hectare of herbicide (chlorotoluron; CHL). (C and D) For studies with fenoxaprop-p-ethyl formulated as Cheetah Super, (C) WTS or (D) MHR Peldon plants were pretreated with NBD-Cl (80 g ai per hectare), before spraying with formulation control (Form) or 85 g ai per hectare of herbicide [fenoxaprop-p-ethyl (FXP)]. (E and F) For studies with the independent MHR Spain black-grass biotype, WTS and MHR Spain black-grass plants were pre-treated with NBD-Cl (270 g ai per hectare) or formulation only followed by a treatment with (E) 165 g ai per hectare of fenoxaprop-p-ethyl (FXP) or formulation only or (F) 250 mL per hectare of clodinafop-propargyl (CDF), as the commercial formulation Topik, or formulation only. In all cases plants were evaluated for phytotoxic injury 21 d postherbicide application.

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Fig. 7.

Nucleophilic aromatic substitution chemistry approaches to analogs explored in this study (EWG, electron withdrawing group; LG, leaving group; Nu, nucleophile).

Plant Studies.

Chemical treatments (herbicides and inhibitors), MHR and WTS black-grass and annual rye-grass and control and transformed lines of Arabidopsis thaliana were prepared as detailed in SI Materials and Methods.

Plant Analysis.

Protein and metabolite analysis and enzyme assays were conducted as described in SI Materials and Methods, using previously described methods. Alterations in the transcriptome of Arabidopsis plants transformed with AmGSTF1 (line 12) relative to untransformed controls were determined in triplicate, using Affymetrix arrays, and the results confirmed by qPCR as described in SI Materials and Methods.

Accessions.

LrGSTF1 nucleotide sequences were deposited with the European Nucleotide Archive (accession no. {"type":"entrez-nucleotide","attrs":{"text":"HF548530","term_id":"461490337","term_text":"HF548530"}}HF548530). Microarray data files have been deposited with the Gene Expression Omnibus (accession no. {"type":"entrez-geo","attrs":{"text":"GSE42065","term_id":"42065"}}GSE42065) (32).

Supplementary Material

Supporting Information:

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^{Department of Chemistry and}

^{School of Biological and Biomedical Sciences, University of Durham, Durham, DH1 3LE, United Kingdom;}

^{Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom;}

^{Biological Sciences, Syngenta, Jealott’s Hill International Research Station, Bracknell, Berks RG42 6EY, United Kingdom; and}

^{Food and Environment Research Agency, Sand Hutton, York YO41 1LZ, United Kingdom}

^{Corresponding author.}

^{To whom correspondence should be addressed. E-mail:}ku.ca.kroy@sdrawde.trebor.

Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved February 26, 2013 (received for review December 6, 2012)

Author contributions: D.H., S.S.K., P.G.S., and R.E. designed research; I.C., D.J.W., F.S., C.R.C., H.E.S., J.D.S., K.K., L.E., and S.-J.H. performed research; I.C., D.J.W., F.S., Z.H., C.R.C., and H.E.S. analyzed data; and I.C., D.J.W., P.G.S., and R.E. wrote the paper.

^{I.C. and D.J.W. contributed equally to this work.}

Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved February 26, 2013 (received for review December 6, 2012)

Abstract

Multiple-herbicide resistance (MHR) in black-grass (Alopecurus myosuroides) and annual rye-grass (Lolium rigidum) is a global problem leading to a loss of chemical weed control in cereal crops. Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to detoxify xenobiotics. In humans, MDR is linked to the overexpression of a pi class glutathione transferase (GSTP1), which has both detoxification and signaling functions in promoting drug resistance. In both annual rye-grass and black-grass, MHR was also associated with the increased expression of an evolutionarily distinct plant phi (F) GSTF1 that had a restricted ability to detoxify herbicides. When the black-grass A. myosuroides (Am) AmGSTF1 was expressed in Arabidopsis thaliana, the transgenic plants acquired resistance to multiple herbicides and showed similar changes in their secondary, xenobiotic, and antioxidant metabolism to those determined in MHR weeds. Transcriptome array experiments showed that these changes in biochemistry were not due to changes in gene expression. Rather, AmGSTF1 exerted a direct regulatory control on metabolism that led to an accumulation of protective flavonoids. Further evidence for a key role for this protein in MHR was obtained by showing that the GSTP1- and MDR-inhibiting pharmacophore 4-chloro-7-nitro-benzoxadiazole was also active toward AmGSTF1 and helped restore herbicide control in MHR black-grass. These studies demonstrate a central role for specific GSTFs in MHR in weeds that has parallels with similar roles for unrelated GSTs in MDR in humans and shows their potential as targets for chemical intervention in resistant weed management.

Abstract

The evolution of herbicide resistance in weeds is a global problem with serious implications to sustainable arable agriculture (1 –3). The best-characterized resistance mechanisms arise from mutations in the proteins targeted by herbicides that lead to a reduced sensitivity to inhibition [target site-based resistance (TSR)]. Mutations leading to TSR have been well described for the plastoquinone-binding protein of photosystem II (PSII) and the acetyl CoA carboxylases (ACCases) and acetolactate synthases involved in fatty acid and branched chain amino acid biosynthesis, respectively (4). Whereas TSR in weeds is widespread, chemical control can be restored by alternating the use of herbicides with differing modes of action (1, 2, 4). A second and more problematic mechanism is based on weeds evolving multiple-herbicide resistance (MHR), which is distinct from herbicide cross-resistance arising from the pyramiding of multiple-TSR traits (4, 5). In MHR, weeds deploy a central defense system, which counteracts herbicide-imposed toxicity irrespective of their mode of action (3). MHR is linked to an enhanced ability of the weeds to detoxify herbicides and has also been termed metabolism-based resistance (3, 6). MHR is most problematic in black-grass (Alopecurus myosuroides) and annual rye-grass (Lolium rigidum), which compete with cereal crops (1). In these weeds an enhanced ability to metabolize herbicides is a powerful route to resistance to graminicides, as the differential rates of detoxification between grasses and cereals represents one of the few biochemical features that can be exploited in selective chemical weed control (6).

MHR was first reported in black-grass in 1982 at Peldon in Essex, England, with independent outbreaks subsequently recorded across Europe (6, 7). Similarly, many populations of MHR annual rye-grass have arisen independently around the world, in some cases pyramiding with TSR traits (8). In grass weeds, MHR is associated with elevated levels of herbicide-detoxifying enzymes, including cytochrome P450 mixed-function oxidases (CYPs), family 1 UDP-glucose-dependent glycosyltransferases (UGTs), and glutathione transferases (GSTs) (9 –11), as well as membrane-associated ATP-binding cassette (ABC) drug transporter proteins (12). Collectively, we have termed these xenobiotic detoxifying enzymes and transporters the “xenome” (13). In MHR black-grass the coordinated up-regulation of the xenome is associated with resistance to several graminicides (14, 15), such as chlorotoluron (PSII-inhibiting phenylurea) and fenoxaprop-p-ethyl (ACCase inhibitor). Previous studies in black-grass identified a specific GST as a highly expressed xenome component in the independent MHR black-grass populations, biotypes “Peldon” and “Spain”, but not in TSR or wild-type sensitive (WTS) plants (14, 15). As a member of the plant-specific phi (F) class of GST (GSTF1), this enzyme has been renamed A. myosuroides (Am) AmGSTF1 (13). Unlike other GSTFs in crops and weeds, AmGSTF1 showed little activity in detoxifying herbicides (15) but was highly active as a glutathione peroxidase (GPOX), catalyzing the reduction of organic hydroperoxides (15). The up-regulation of AmGSTF1 in MHR black-grass shows several intriguing parallels with the enhanced expression of unrelated pi (P) class GSTs (GSTPs) in multiple-drug-resistant (MDR) tumors in humans. In addition to directly detoxifying therapeutic drugs and quenching the formation of toxic hydroperoxides formed during treatment, GSTPs directly regulate signaling pathways that promote cellular defense (16). On the basis of this precedence for a GST to orchestrate MDR in humans, we have investigated the potential for the unrelated GSTF1s to have a regulatory role in MHR in weeds, through a combination of transgenesis and chemical inhibition studies.

Measurements are shown ± SEM, n = 3. FW: fresh weight.

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Acknowledgments

This work was supported by joint funding from the United Kingdom’s Biotechnology and Biological Sciences Research Council and Syngenta (Grant BB/G006474/2).

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences and data reported in this paper have been deposited in the European Nucleotide Archive (accession no. {"type":"entrez-nucleotide","attrs":{"text":"HF548530","term_id":"461490337","term_text":"HF548530"}}HF548530) and the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. {"type":"entrez-geo","attrs":{"text":"GSE42065","term_id":"42065","extlink":"1"}}GSE42065).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1221179110/-/DCSupplemental.

Footnotes

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