Pesticide Biochemistry and Physiology 84 (2006) 227–235

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PESTICIDEBiochemistry & Physiology

Multiple effects of a naturally occurring proline to threoninesubstitution within acetolactate synthase in two

herbicide-resistant populations of Lactuca serriola

Christopher Preston a,c,*, Lynley M. Stone a,b,1, Mary A. Rieger a,b,Jeanine Baker a,c

a Cooperative Research Centre for Australian Weed Management (formerly Cooperative Research Centre for Weed

Management Systems), PMB 1, Glen Osmond, SA 5064, Australiab Department of Applied and Molecular Ecology, Waite Campus, University of Adelaide, PMB 1, Glen Osmond, SA 5064, Australia

c School of Agriculture and Wine, Waite Campus, University of Adelaide, PMB 1, Glen Osmond, SA 5064, Australia

Received 8 March 2005; accepted 26 July 2005Available online 21 October 2005

Abstract

Two populations of Lactuca serriola L. with resistance to acetolactate synthase (ALS)-inhibiting herbicides were dis-covered in wheat fields at two locations more than 25 km apart in South Australia. Both resistant populations carried asingle base change within a highly conserved coding region of the ALS gene that coded for a single amino acid mod-ification within ALS. The modification of proline 197 to threonine resulted in an enzyme that was highly resistant(>200-fold) to inhibition by sulfonylurea herbicides and moderately resistant to triazolopyrimidine and imidazolinoneherbicides. The herbicide-resistant ALS was also less sensitive to inhibition by the branched-chain amino acids valineand leucine. In addition, the resistant enzyme had a lower Km for pyruvate. However, extractable ALS activity was sim-ilar between resistant and susceptible plants. The substitution of threonine for proline 197 within ALS has multipleimpacts on ALS enzyme activity in L. serriola that may influence the frequency of this resistant allele in theenvironment.� 2005 Elsevier Inc. All rights reserved.

Keywords: Acetohydroxyacid synthase; Prickly lettuce; Chlorsulfuron; Valine; Leucine

0048-3575/$ - see front matter � 2005 Elsevier Inc. All rights reserve

doi:10.1016/j.pestbp.2005.07.007

* Corresponding author. Fax: +08 8379 4095.E-mail address: christopher.preston@adelaide.edu.au

(C. Preston).1 Present address: Department of Conservation and Land

Management, Locked Bag 104, Bentley Delivery Centre, WA6983, Australia.

1. Introduction

The sulfonylurea, imidazolinone and triazolo-pyrimidine herbicides are widely used to controlweeds in numerous crops across southernAustralia.

d.

228 C. Preston et al. / Pesticide Biochemistry and Physiology 84 (2006) 227–235

These herbicides are potent inhibitors of the plas-tidic enzyme acetolactate synthase or acetohydr-oxyacid synthase (EC 2.2.1.6 ALS or AHAS).This enzyme catalyzes two reactions: the conden-sation of two molecules of pyruvate tosynthesize acetolactate and the condensation ofpyruvate and 2-ketobutyrate to form 2-acet-ohydroxybutyrate [1]. ALS is the first step in thepathway for the synthesis of the branched-chainamino acids, leucine, isoleucine, and valine andis feedback inhibited by these amino acids [2].One unfortunate consequence of the widespreadadoption of sulfonylurea herbicides has been theappearance of weed populations with resistanceto ALS-inhibiting herbicides. Resistance has nowbeen documented for populations of a numberof weed species [3].

Amino acid substitutions at 10 different siteswithin the yeast ALS protein are known to conferresistance to one or more class of ALS-inhibitingherbicide [4]. However, in higher plants, aminoacid substitutions that confer resistance have beenobserved at five different sites in laboratory-gener-ated and field mutants [3]. Each site is locatedwithin a highly conserved region of the ALS en-zyme. Ala 122 occurs within a conserved regionconsisting of 18 amino acids and when substitutedby Thr resistance to imidazolinone herbicides oc-curs [5]. Pro 197 occurs within a conserved regionof 13 amino acids [6]. Substitution of any one of anumber of amino acids for Pro results in an ALSwith resistance to sulfonylurea and triazolopyrim-idine herbicides and, to a lesser extent, to imidaz-olinone herbicides [3,7]. Ala 205 occurs within aconserved sequence of six amino acids and whensubstituted by Val resistance to sulfonylurea andimidazolinone herbicides occurs [8]. Trp 574 oc-curs within a conserved sequence of four aminoacids. Substitution of Leu for Trp confers resis-tance to all three of the above chemical classes[9]. Lastly, Ser 653 occurs within a conserved re-gion of six amino acids. Substitution of eitherAsp or Thr for Ser at this position confers resis-tance to imidazolinone herbicides [10,11]. Certainmutations within ALS are more commonly report-ed in weed species selected in the field than areother possible mutations [3]. Why this should beis unclear, but probably relates to differential

fitness of the different mutations. For this to occur,mutations within ALS must have other effects onenzyme function. Understanding how naturallyoccurring modifications of ALS affect enzymefunction may provide insights into the evolution-ary processes that keep these mutations rare inunselected populations, but not so rare that theyare rapidly selected by herbicide use.

Lactuca serriola is not normally considered aserious weed of southern Australian agricultureas it tends to germinate in early spring and pro-vides minimum competition for crops. WhileL. serriola is not normally a major target of herbi-cides in crops, in 1995, a population of L. serriolawas reported resistant to sulfonylurea herbicidesfollowing four applications of these herbicidesover 6 years to the wheat field in which it wasgrowing. A second population was discovered inanother wheat field more than 25 km away in1997. This study examines properties of ALS fromthese resistant populations and compares them tothe enzyme from two susceptible populations.

2. Materials and methods

2.1. Plant material

Two resistant populations of L. serriola L. werecollected. The first (SLS1) was collected from awheat crop near Bute, SA. This field had receivedfour applications of chlorsulfuron or triasulfuronbetween 1989 and 1995. Following the failure oftriasulfuron to control L. serriola in 1995, strips ofthe field were treated with sulfometuron–methyl in1996, which also failed to control this population.Seeds were collected from survivors of the 1996treatments. Seed of the second resistant population(SLS3) was collected from individuals surviving in awheat field near Kulpara, SA, after harvest in 1997.The herbicide history of this field is unknown, but ispresumed to include exposure to sulfonylurea herbi-cides as these herbicides are widely used on wheatfields in this area. Seed of the two susceptible popu-lations (SLS2 and SLS4) were collected from twodifferent sites, one a reserve and one waste ground,around Adelaide, SA. Neither of these sites hadany known history of ALS-inhibiting herbicide

C. Preston et al. / Pesticide Biochemistry and Physiology 84 (2006) 227–235 229

use. Seedlings of the four populations had slight dif-ferences in leaf morphology.

Seeds were sown on 0.6% (w/v) agar and placedat 4 �C in the dark for 24 h. After this chillingtreatment, seeds were placed in a germination cab-inet with a 12 h, 19 �C, 30 lmol m�2 s�1 light peri-od and a 12 h, 19 �C dark period for 4 days togerminate. Seedlings were then transplanted topotting soil.

2.2. Genomic DNA extraction, PCR amplification,

and sequencing of partial ALS gene

About 1 g of young leaf tissue was collectedfrom either resistant or susceptible plants and fro-zen immediately in liquid N2. DNA was extractedusing the method of Doyle and Doyle [12]. Thepelleted DNA was re-suspended in 400 lL 10mM EDTA, 10 mM Tris–HCl (pH 7.4), RNAwas removed with 1% (v/v) RNase and the DNAstored at 4 �C.

A 196 bp fragment of the L. serriola ALS genewas amplified by PCR as described by Guttieri andEberlein [13] using their primers 1 and 4. The PCRmixture contained 0.1 lM of each primer, 200 lMof each dNTP, 5 lL of 10· thermophilic buffer(Promega, USA), and 1 lL DNA sample in a finalvolume of 50 lL. Amplifications were carried outin a heated lid PCR machine (PTC-100, MJ Re-search) where the DNA was denatured at 94 �Cfor 8.5 min. At this point, 2.5 U of TaqDNA poly-merase (proofreading, Promega, USA) were add-ed. PCR conditions were 1.5 min at 94 �C, 2 minat 50 �C, and a 2 min at 72 �C. The cycle wasrepeated 34 times with a final elongation step at72 �C for 10 min.

The PCR products were separated on a 2% aga-rose gel and a band of about 196 bp was present ineach sample. This band was excised from ethidiumbromide-stained gels and purified using a WizardPCR preps DNA purification kit according tomanufacturers specifications (Promega, USA).

The purified PCR product was sequenced withan Applied Biosystems Model 373A DNAsequencer using a PRISM Ready Reaction Dye-Deoxy Terminator Cycle Sequencing kit (AppliedBiosystems). The DNA samples were sequencedin both directions, twice.

2.3. ALS isolation and assay

Plants were grown in potting soil in 30 · 40 cmtrays with 50 plants per tray in a growth room setat 12 h, 20 �C, 400 lmol m�2 s�1 light period and a12 h, 15 �C dark period. As a result of interferencecaused by the milky latex in L. serriola leaves, ALSwas extracted from leaf tissue by first isolatingchloroplasts. Chloroplasts were rapidly isolatedfrom 15 g of leaves of resistant or susceptible bio-types by the method of Cerovic and Plesnicar [14],without the subsequent washing steps. The chloro-plasts were suspended in 5 mL of 0.2 M potassiumphosphate buffer (pH 7.0), 20 mM MgCl2,200 mM sodium pyruvate, 2 mM thiamine diphos-phate, and 20 lM flavine adenine nucleotide. Thesuspension was placed in a sonicator bath for30 s and then centrifuged at 25,000g for 10 minto pellet thylakoids and chloroplast envelopes.The resulting supernatant was used immediatelyas a crude extract for enzyme assays. All steps, ex-cept the sonication were conducted at 4 �C. Thisextraction procedure provided a rapid, less than25 min, and repeatable method of measuringALS from species such as L. serriola.

Enzyme assays were conducted in 400 lL wellmicro assay plates (Labsystems) using a modifica-tion of the procedure described by Ray [15]. Ineach well, 50 lL of water or water containing theappropriate inhibitor solution was placed and theplates incubated at 37 �C. The inhibitors used weretechnical grade herbicides chlorsulfuron, triasulfu-ron, sulfometuron–methyl, flumetsulam, imazapyrand imazethapyr, and the amino acids valine andleucine. The reaction was initiated by the additionof 50 lL of crude enzyme extract and incubatedfor 30 min at 37 �C. The reaction was terminatedby addition of 20 lL 6 N H2SO4 and the solutionheated for 15 min at 60 �C to decarboxylate aceto-lactate to acetoin. The resulting acetoin was react-ed with 95 lL of 0.55% creatine and 95 lL of 5.5%a-naphthol (in 5 N NaOH) for 15 min at 60 �C todevelop color. Following color development, theabsorbance was measured at 530 nm in a spectro-photometer. Background color development wasmonitored by the addition of 6 N H2SO4 prior toaddition of the enzyme extract and this was sub-tracted from all measurements. Enzyme activity

230 C. Preston et al. / Pesticide Biochemistry and Physiology 84 (2006) 227–235

was calculated as a percentage of activity in the ab-sence of inhibitor and I50 concentrations for herbi-cides calculated following log sigmoidaltransformation of the data. For feedback inhibi-tion by amino acids, curves were fitted to the fol-lowing equation by non-linear regression analysis(Prism, GraphPad Software):

y ¼ Aþ ð100� AÞ=ð1þ ½amino acid�=K iÞ;

where A is the residual activity (in %) at an infiniteconcentration of amino acid inhibitor and Ki is theapparent inhibition constant.

For measurements of Km(pyruvate) and Vmax,the chloroplasts were re-suspended in 5 mL of0.2 M potassium phosphate buffer (pH 7.0),20 mM MgCl2, 2 mM sodium pyruvate, 2 mM thi-amine diphosphate, and 20 lM flavine adeninenucleotide. The assays were conducted as de-scribed above in the absence of herbicide, but withvarying concentrations of sodium pyruvate. Vmax

and Km(pyruvate) were calculated by fitting the re-sults to the following equation by non-linearregression (Prism, GraphPad Software):

v ¼ V max=ð1þ KmðpyruvateÞ=½pyruvate�Þ;where v is the reaction velocity at any concentra-tion of pyruvate.

The assays to measure the inhibitory effect ofherbicides on ALS were conducted with two pop-ulations, one resistant and one susceptible, at atime. The assays to measure the inhibitory effectsof valine and leucine were conducted with all fourpopulations, as were the assays to measureKm(pyruvate) and Vmax. Each set of assays wasconducted on three separate enzyme preparations.

3. Results and discussion

3.1. Whole plant dose–response to herbicides

Experiments using whole plants showed thatthe two populations of L. serriola from wheatfields were resistant to the sulfonylurea herbicideschlorsulfuron and sulfometuron–methyl (data notshown). The two susceptible populations were eas-ily controlled by low rates of these herbicide withalmost no survival at 0.3 g ha�1 chlorsulfuron.

In contrast, the two resistant populations werenot controlled by 64 g ha�1 chlorsulfuron. L. serri-ola population SLS1 was also highly resistant tometsulfuron–methyl and triasulfuron. This popu-lation also showed substantial, although, lowerresistance to imazapyr and imazethapyr (data notshown). Many examples of plants with resistanceto ALS-inhibiting herbicides are known and thepatterns of resistance across herbicide chemicalclasses can vary [3,7]. A L. serriola population withresistance to chlorsulfuron was discovered in Ida-ho in 1989 [16]. This population was also highlyresistant to chlorsulfuron with moderate resistanceto the imidazolinone herbicides.

Resistance to sulfonylurea herbicides in L. serri-

ola was selected rapidly with only four applica-tions of sulfonylurea herbicides. There are likelyto be several factors that have contributed to therapid selection of resistance to these herbicides.First, resistance to sulfonylurea herbicides is pres-ent at relatively high frequency in untreated weedpopulations [17], allowing resistance to be rapidlyselected once herbicides are used. Second, the sus-ceptible populations of L. serriola are controlledby very low concentrations of sulfonylurea herbi-cides, much lower than normal field rates. Third,the area where the resistant populations occurredis characterized by alkaline soils, which allow sul-fonylureas to persist for a significant period oftime [18].

3.2. PCR amplification and identification of

mutations in the ALS gene

The majority of examples of resistance to ALS-inhibiting herbicides are the result of amino acidchanges within the target enzyme ALS [9]. Manyof these carry an amino acid modification at Pro197 within a highly conserved region of the ALS[19]. This region of the ALS gene was amplifiedby PCR and the 196 bp fragment obtained se-quenced for both resistant and susceptible popula-tions. The nucleotide sequence from all fourL. serriola populations was nearly identical overthis region. There was a single change, cytosineto adenine, present in both resistant populations.This nucleotide change encoded an amino acidchange from Pro to Thr within this highly

C. Preston et al. / Pesticide Biochemistry and Physiology 84 (2006) 227–235 231

conserved region. The two resistant L. serriola

populations had the same amino acid modificationdespite being collected more than 25 km apart. L.serriola is a self-pollinated species with wind-bornseed. Therefore, it is possible that the two popula-tions had the same origin. However, the differencesin leaf morphology between the different popula-tions suggest they have different origins.

3.3. ALS response to herbicides, substrates, and

feedback inhibitors in vitro

The ALS extracted from chloroplasts isolatedfrom leaves of resistant and susceptible plantswas resistant to ALS-inhibiting herbicides. Chlor-sulfuron inhibited activity of ALS from both resis-tant and susceptible plants (Fig. 1); however, morethan 100 times the concentration was required toinhibit the enzyme from resistant plants comparedto susceptible plants. There were only minor differ-ences in susceptibility of the enzyme from suscep-tible plants with activity decreased to less than20% of the control with 1 lM chlorsulfuron. Incontrast, 1 lM chlorsulfuron resulted in a smallinhibition of activity of ALS from either of theresistant populations. I50 values for each herbicideand population were calculated from the log sig-moidal lines fitted to the data. The susceptiblepopulations had similar I50 values ranging from

100

80

60

40

20

00.0001 0.001 0.01 0.1 1 10 100

Chlorsulfuron concentration (µM)

ALS

act

ivit

y (%

con

trol

)

Fig. 1. Chlorsulfuron inhibition of ALS activity isolated fromchloroplasts of susceptible, SLS2 (h) and SLS4 (s), andresistant, SLS1 (j) and SLS3 (d), populations of Lactuca

serriola. Each point is the mean ± SE of three replicateexperiments each assayed in duplicate.

40 nM for chlorsulfuron to 10 lM for imazapyrdespite being assayed in separate experiments(Table 1). Both resistant populations were resis-tant to all six ALS-inhibiting herbicides tested withI50 values ranging from 1 lM for flumetsulam to110 lM for imazapyr. The resistant populationswere highly resistant (>200-fold) to all the sulfo-nylurea herbicides and about 10-fold resistant toflumetsulam and the imidazolinone herbicides.The I50 values of the two resistant populationswere similar.

ALS is a key enzyme in branched-chain aminoacid biosynthesis in plants and a target for fourdifferent herbicide chemistries. Modifications toALS resulting in resistance to herbicides occurreadily and have been selected in tissue cultureand other laboratory selection systems [19] as wellas in the field [20]. Mutations within ALS resultingin resistance to herbicides are known at five differ-ent sites in plants [9]. One of the most common ofthese mutations is at Pro 197 [3,19]. It appears thatany amino acid substitution at this proline will re-sult in an active enzyme that is resistant to herbi-cides [21]. The Thr for Pro 197 substitutionfound in both resistant L. serriola populationshas been observed previously in resistant popula-tions of Kochia scoparia [21], Raphanus raphani-

strum [22], and Papaver rhoeas [23], and thereforeappears to be a common substitution.

Most amino acid substitutions at Pro 197 resultin an enzyme with high resistance to sulfonylureaherbicides, lower resistance to flumetsulam andvarying resistance to imidazolinone herbicides [7].The Pro 197 to Thr mutation in L. serriola resultedin an enzyme with very high resistance to sulfonyl-urea herbicides, particularly sulfometuron–methyl,and moderate resistance to both triazolopyrimi-dine and imidazolinone herbicides (Table 1). Apopulation of L. serriola from Idaho with a Pro197 to His substitution shows high resistance tochlorsulfuron, moderate resistance to imazethapyrand low resistance to flumetsulam [24]. Clearly,different modifications at the Pro 197 can resultin variation in resistance across herbicide chemicalclasses.

ALS is feedback inhibited by branched-chainamino acids [25]. Both valine and leucine inhibitedthe enzyme isolated from susceptible plants

Table 1I50 values and resistance ratios for ALS inhibitor herbicides using ALS isolated from chloroplasts of susceptible or resistantpopulations of L. serriola

Herbicide SLS1 (R) (lM) SLS2 (S) (lM) R/S SLS3 (R) (lM) SLS4 (S) (lM) R/S

Chlorsulfuron 8.39 ± 1.4 0.04 ± 0.02 210 9.43 ± 0.72 0.04 ± 0.01 236Triasulfuron 26.9 ± 6.6 0.10 ± 0.03 269 25.7 ± 3.8 0.09 ± 0.02 285Sulfometuron–methyl >100 0.19 ± 0.03 >500 >100 0.16 ± 0.04 >500Flumetsulam 1.45 ± 0.26 0.13 ± 0.05 11 1.22 ± 0.12 0.10 ± 0.03 12Imazapyr 115 ± 11.8 10.5 ± 1.8 11 109 ± 10.0 9.20 ± 1.5 12Imazethapyr 62.6 ± 10.9 9.75 ± 1.7 6 34.5 ± 2.2 7.06 ± 1.5 5

Values are means (±SE) from three extractions each assayed in duplicate.

232 C. Preston et al. / Pesticide Biochemistry and Physiology 84 (2006) 227–235

(Fig. 2). Valine inhibited ALS activity by between47 and 57% whereas leucine inhibited ALS activityby between 63 and 68%. In contrast, branched-chain amino acids were less effective at inhibitingALS activity from resistant plants. Leucine inhib-ited ALS activity from the resistant plants by 47%,while valine inhibited activity by between 19 and24%. The ALS enzyme from the two resistant pop-ulations behaved similarly. Ki values tended to beslightly greater for the resistant populations thanfor susceptible populations for both amino acids.However, the differences may not be significant.

It has been previously observed that a herbi-cide-resistant ALS enzymes are less sensitive tofeedback inhibition by valine and leucine [24,26–28]. An ALS enzyme carrying an amino acid mod-ification at Pro 197 was inhibited by both valineand leucine [24]. In contrast, mutations in other re-

0 1 2 3 4 50

20

40

60

80

100 A

Valine concentration (mM)

AL

Sac

tivi

ty(%

cont

rol)

Fig. 2. Inhibition of ALS isolated from susceptible, SLS2 (h) and SLLactuca serriola by valine (A) and leucine (B). Each point is the meanEstimated values (±SE) for A and Ki from the fitted curves are for v0.45 ± 0.15 mM; SLS1, 76.3 ± 2.3%, 0.49 ± 0.19 mM; and SLS3, 80.0.10 ± 0.01 mM; SLS4, 31.6 ± 2.8%, 0.14 ± 0.03 mM; SLS1, 53.1 ± 1

gions of ALS do not appear to alter feedback inhi-bition by valine [29]. Valine resistance is alsoconferred by a Ser 214 to Leu change in tobaccoALS, a modification that does not result in herbi-cide-resistance [30]. Typically, leucine is a moreeffective inhibitor of ALS than is valine [31]. Thesame is true for the susceptible and resistant pop-ulations of L. serriola examined here (Fig. 2).However, in a population of L. serriola with aPro 197 to His substitution, valine was a moreeffective inhibitor of ALS than leucine [24]. Thisclearly demonstrates different mutations Pro 197result in differences in the ability of branched-chain amino acids to act as feedback inhibitors.

The regulatory subunit of ALS is responsiblefor feedback inhibition by branched-chain aminoacids in Arabidopsis thaliana [32,33]. As the regula-tory subunit apparently contains separate sites for

0 1 2 3 4 5

B

Leucine concentration (mM)

S4 (s), and resistant, SLS1 (j) and SLS3 (d), populations of± SE of three replicate experiments each assayed in duplicate.aline: SLS2, 53.5 ± 1.7%, 0.23 ± 0.04 mM; SLS4, 42.9 ± 5.3%,9 ± 4.3%, 0.54 ± 0.18 mM and for leucine: SLS2, 31.6 ± 2.8%,.7%, 0.18 ± 0.03 mM; and SLS3, 52.9 ± 2.4%, 0.20 ± 0.05 mM.

C. Preston et al. / Pesticide Biochemistry and Physiology 84 (2006) 227–235 233

interaction for valine and leucine [33], it is perhapssurprising that mutations on the catalytic subunitsshould influence feedback inhibition. One possibil-ity is through conformational changes on the cat-alytic subunit, which affect either binding of theregulatory unit or transmission of the inhibitorysignals to the catalytic subunit. For example, Linet al. [34] have proposed that a Gln substitutionfor Pro in ALS from Limnophila sessiflora createda local change in secondary structure from extend-ed strands to an a-helix. What is not clear is whydifferent substitutions at Pro 197 will result in dif-ferences in feedback inhibition. Clearly, feedbackinhibition in ALS is a complex process.

The maximum rate of ALS activity was slightlyreduced in resistant compared to susceptible popu-lations (Table 2); however, these differences werenot consistent across populations. On some occa-sions, variations in specific activity of ALS havebeen observed with resistant ALS enzymes. Forexample, two populations of Sisymbrium orientale

from South Australia had higher extractable ALSactivity than a susceptible population or anotherresistant population [35]. Also a population ofL. serriola from Idaho had lower specific activitythan a susceptible population [24]. Site-directedmutations of ALS frequently result in lower or asimilar level of extractable activity [36,37]. It isnot known whether such small differences in totalspecific activity are important. In contrast, tomany other studies of herbicide-resistant ALS,resistant populations of L. serriola had an ALSthat displayed a difference in Km(pyruvate). Thecalculated Km(pyruvate) for the susceptible popu-lations was about 10 mM, whereas that for theresistant populations was about 5 mM (Table 2).

Table 2Km(pyruvate) and specific activity of ALS extracted fromchloroplasts of susceptible and resistant populations of L.

serriola

Population Km(pyruvate)(mM)

Specific activity (nmol min�1

mg�1 protein)

SLS1 (R) 5.4 ± 0.1 8.0 ± 0.3SLS2 (S) 10.0 ± 0.6 9.9 ± 0.2SLS3 (R) 4.9 ± 0.4 9.7 ± 0.2SLS4 (S) 9.7 ± 0.6 12.2 ± 0.2

Values are means (±SE) from three extractions each assayed induplicate.

Some site directed mutations of Ser 653 also resultin an enzyme with reduced Km(pyruvate) [38].Again it is not known what significance a reducedKm(pyruvate) would have; however, these resultsdemonstrate that some amino acid substitutionsin ALS result in changes to enzyme function aswell as insensitivity to herbicides.

The target site mutation of Ser 196 to Gly in theD1 protein of photosystem II is well known to re-sult in a fitness penalty in resistant plants as a re-sult of a significant decrease in photosyntheticelectron transport [39]. In PS II, Ser 196 plays acrucial role in the binding of herbicides like atra-zine and an important role in stabilizing electrontransport to plastoquinone [40]. In the case ofALS, the impact of mutations on enzyme functionand hence ecological fitness are less clear. Panget al. [41] have shown that Pro 197 is part of thesubstrate access channel in the yeast ALS enzymeand has direct hydrophobic interactions with thephenyl ring of sulfonyurea herbicides. Many sub-stitutions at Pro 197 are possible and all result inan active enzyme resistant to herbicides [3,21]. Incontrast, very few amino acid substitutions arepossible at Trp 591 in another highly conserved re-gion of ALS [5], and only one has been found tooccur naturally. This might suggest that Pro 197is not directly involved in the catalytic activity ofALS; however, changes at this residue can havedramatic effects on enzyme function including, de-creased sensitivity to herbicides, decreased sensi-tivity to feedback inhibition by branched-chainamino acids and changes to enzyme activity. Dif-ferent amino acid substitutions at Pro 197 appearto result in an enzyme with different characteris-tics. The results here show that the Pro 197 toThr change has little effect on the ability of ALSto synthesize acetolactate. This raises the questionof why Pro is preferred at this site to other aminoacids in wild-type plants. The answer probably liesin the reduced feedback inhibition in mutants ofPro 197 by the amino acids valine and leucine. Areduction in feedback inhibition will mean thatresistant plants will produce more branched-chainamino acids. This may upset the amino acid poolsin the plants and provide the fitness penalty thatmaintains the mutant alleles at low frequencies inuntreated populations.

234 C. Preston et al. / Pesticide Biochemistry and Physiology 84 (2006) 227–235

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