Zhuo Chen,Zheng Wng,Ynfng Heng,Jin Li,Jiwei Pei,Ying Co,Xing Wng Deng,*,Ligeng M,*

a College of Life Sciences,Beijing Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement,Capital Normal University,Beijing 100048,China

b School of Advanced Agricultural Sciences,Peking University,Weifang 261000,Shandong,China

Keywords:Wheat Herbicide Genetic screening Imidazolinone resistance Taals alleles

ABSTRACT The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively.Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals,are effective at small doses,and exhibit broad-spectrum herbicidal action in the field.Therefore,the isolation and genetic and molecular characterization of IMI-resistant wheat mutants will enhance weed management in wheat fields.In the present study,352 IMI-resistant plants were isolated by genetic screening from a mutant pool prepared by EMS-based random mutagenesis.Cloning of the mutated genes from the IMI-resistant plants indicated that ten taals alleles had been isolated,and mutation in one of three TaALS homolog genes conferred IMI resistance,and such a mutation is a dominant trait.Further analysis showed that taals-d exhibited the greatest IMI resistance,whereas taals-b exhibited the weakest resistance to IMI among three homologous taals mutants.In terms of IMI resistance,the taals triple mutant was stronger than the taals double mutants,and the taals double mutants were stronger than the single mutants,indicating a dose-dependent effect of the TaALS mutation on IMI resistance in wheat.Biochemical analysis indicated that the mutation in TaALS increased the tolerance of TaALS to inhibition by IMI.Our work details the genetic and molecular characterization of als wheat mutants,provides a foundation for understanding IMI resistance and breeding wheat varieties with herbicide resistance,and indicates that genetic screening using a mutagenized pool is an effective and important means of breeding crops with additional desired agricultural traits.

1.Introduction

Bread wheat(Triticum aestivumL.;2n=6x=42;AABBDD)is one of the most important food crops worldwide;it provides calories,proteins,vitamins,minerals,and other nutrients needed by humans [1–3].However,weeds in wheat fields can compete with wheat plants for growth factors,leading to reduced yields and product quality losses[4,5].The control of weeds in crop fields by herbicides is both economical and effective.Imidazolinone (IMI)herbicides are broad spectrum,require low application rates,and are mammal-friendly;therefore,IMI herbicides are among the most widely used herbicides in the field [6–8].However,both weeds and wheat plants are sensitive to IMI herbicides;as a result,their application for weed control in wheat fields is limited[9,10].Therefore,the isolation and breeding of IMI-resistant wheat varieties is key to controlling weeds in wheat fields using IMI herbicides.

The activity of acetolactate synthase (ALS;EC 2.2.1.6),the first common enzyme in valine,leucine,and isoleucine biosynthesis,is inhibited by IMI herbicides [11–15].In the pathway leading to valine and leucine biosynthesis,two pyruvate molecules are condensed to form acetolactate;pyruvate and α-ketobutyrate then undergo decarboxylation to acetohydroxybutyrate,enabling the production of isoleucine [16,17].Early studies in plants indicated that ALS is a chloroplast-localized holoenzyme with four catalytic and four regulatory subunits encoded by nuclear genes [18,19].The IMIs absorbed by plants bind to specific sites in ALS and block the substrate access channel,causing a deficiency in branchedchain amino acids,and eventual plant death [20,21].

The mutation of ALS may reduce its sensitivity to IMIs without obviously decreasing its catalytic activity.Thus,plants that are resistant to IMI herbicides may be generated by the mutation of ALS[22–24].Most IMI-resistantArabidopsismutants carry a substitution for amino acid residues A122,A205,W574,or S653 in ALS[16].TheArabidopsismutantscsr 1–2(S653N in AtALS),csr 1–5(A122T in AtALS),andcsr 1–6(A205V in AtALS) all exhibit resistance to IMI herbicides [25].W542L in ZmALS2 and S621N in ZmALS1 can both confer IMI resistance to maize (Zea maysL.)[26].Resistance to IMIs has also been documented in feral radish(Raphanus sativusL.) due to the substitution W560L in ALS [27].Additional species in which IMI resistance was generated by the mutation of ALS have been reviewed [12].In wheat,Newhouse et al.[9,10] identified an IMI-resistant line of the French winter wheat cultivar ‘Fidel’ carrying a S627N substitution in TaALS-D,while Li et al.[28]identified an IMI-resistant line of the Australian wheat cultivar ‘Brookton’ carrying an A96T substitution in TaALSD.However,detailed genetic and molecular characterizations and studies of the mechanism of IMI resistance inalswheat mutants are needed.

Here,we isolated IMI-resistant mutants of common wheat from mutagenized seed pools prepared by ethyl methanesulfonate(EMS) mutagenesis.Detailed phenotypic,genetic,and molecular characterizations of thetaalsmutants revealed that the substitution A96T,P171L,A179V,or S627N (corresponding to TaALS-D)at one of threeTaALSsconferred resistance to IMI herbicides.taals-dexhibited the strongest IMI resistance among the three homologoustaalsmutants.The IMI resistance of thetaalstriple mutant was greater than that of thetaalsdouble mutants,and thetaalsdouble mutants showed greater IMI resistance than thetaalssingle mutants,indicating that the IMI resistance of thetaalsmutants increased in a dose-dependent manner.In vitroenzymatic assays showed that mutated TaALS was less sensitive to IMI inhibition than wild-type TaALS.Therefore,our work details the genetic characterization and underlying mechanism of IMI resistance in hexaploid wheat mutants and provides a foundation for breeding wheat varieties with herbicide resistance.

2.Materials and methods

2.1.Plant materials

Common wheat(T.aestivumL.)was used in this study.The IMIresistant lines were identified from an EMS-mutagenized population of common wheat (varieties ‘Ningchun 4’,‘Aikang 58’,‘Lunxuan 987’,‘Zhoumai 16’,and ‘Jing RS801’).All plants were grown in a greenhouse under 16 h of light(24°C,at a white light intensity of 250 μmol m-2s-1) and 8 h of darkness (19 °C).

2.2.Preparation of the EMS-mutagenized population and isolation of IMI-resistant lines

The EMS-mutagenized populations were prepared as described[29,30].Seeds of common wheat from different varieties were treated with 0.5% (v/v) EMS (Sigma-Aldrich,St.Louis,MO,USA)for 12 h at～ 25 °C,and the M1population was planted in Yunnan,China.M2seeds were soaked in a 0.5% (v/v) commercial IMI herbicide (～1.7 mmol L-1imazethapyr;Cynda Chemical Co.,Ltd.,Shandong,China) for 18 h before being grown in a growth chamber.IMI-resistant plants were identified after 7–10 days of growth.They were planted in soil until harvest after recovering.In total,352 IMI-resistant mutant plants were isolated.On average,1 resistant mutant plant was identified for every 20,000 M2seeds.

2.3.Genomic DNA extraction,PCR amplification,and sequence analyses

DNA was extracted from 2-week-old plants by the CTAB method [31].PCR was carried out in 50-μL reaction mixtures containing 500 ng of template DNA,0.4 μmol L-1primers,300 μmol L-1dNTPs,1× PCR buffer,and 1 U of KOD-FX DNA polymerase(Toyobo Co.,Ltd.,Osaka,Japan).DNA from the gel was collected with a HiPure Gel Pure Micro Kit (Magen,Guangdong,China).Alignment of the DNA or protein sequences was performed after sequencing.The primers used for PCR are given in Table S1.

2.4.Characterization of herbicide tolerance

After the identification of IMI-resistant plants carrying S627N(corresponding to TaALS-D)at each TaALS,we crossed them to prepare higher-ordertaalsmutants.Seeds from ‘Ningchun 4′,taals-a,taals-b,taals-d,taals-ab,taals-ad,taals-bd,andtaals-abdwere placed on 9-cm Petri dishes after soaking with a different herbicide at each concentration for 18 h.The germinated seeds were transferred to a growth chamber (16 h light/22 °C,8 h dark/22 °C,at a white light intensity of 150 μmol L-1m-2s-1)after 3 days of treatment at 4 °C.A phenotypic analysis was performed after 4 days of treatment.The concentrations of herbicide used for phenotypic analysis are listed below:imazethapyr was at 9.375,37.5,150,600,1200,2400,and 4800 μmol L-1,respectively;imazamox was at 0.6,2.4,and 4.8 mmol L-1,respectively;imazapic was at 0.6,2.4,and 4.8 mmol L-1,respectively;mesosulfuron-methyl was at 0.625,2.5,10,and 40 μmol L-1,respectively;flumetsulam was at 75,150,300,and 1200 μmol L-1,respectively;pyribenzoxim was at 10,40,160,and 320 μmol L-1,respectively;flucarbazonesodium was at 20,80,320,and 1280 μmol L-1,respectively.The reported mean fresh weights and dry weights (% of control) are from three independent biological replicates,and 30 plants are used in each biological replicate of each treatment(each herbicide at each concentration).

2.5.Expression and purification of TaALS-His fusion proteins using E.coli

TaALS-His fusion proteins were expressed and purified as reported previously [2,32].Genomic DNA from ‘Ningchun 4′or thetaalsmutants were used to amplify the different truncated TaALS (lacking the chloroplast transit peptide predicted by ChloroP;http://www.cbs.dtu.dk/services/ChloroP/).Each PCR product was fused intopET-28a(+) digested withEcoR I andNdeI.Escherichia coli(strain BL21[DE3])cells containing the appropriate plasmid were grown in 4 mL of 2× YT medium overnight to produce a 200 mL culture.Next,0.1 mmol L-1IPTG was added to induce target protein expression at an A600of 0.8–1.0,and the bacteria were incubated for 18 h at 16 °C.

The cells were collected and resuspended in lysis buffer(50 mmol L-1NaH2PO4,300 mmol L-1NaCl,10 mmol L-1imidazole,5 mmol L-1MgCl2,1 mmol L-1EDTA-Na2,1 mg mL-1of lysozyme,and 10% [v/v] glycerin,pH 7.0).After incubation on ice for 30 min,the cells were lysed by sonication and cell debris was removed by centrifugation at 20,000×g.Ni-NTA agarose (Qiagen,Inc.,Hilden,Germany)was used for protein purification.Lysis buffer containing 20 mmol L-1imidazole was used to free proteins bound nonspecifically with the agarose.Each target fusion protein was eluted with elution buffer (10 mmol L-1NaH2PO4,150 mmol L-1NaCl,300 mmol L-1imidazole,5 mmol L-1MgCl2,1 mmol L-1EDTA-Na2,and 10% [v/v] glycerin,pH 7.0).The protein samples were frozen in liquid nitrogen immediately and stored at -80 °C until the enzymatic assay.The protein concentration was determined using Bradford Reagent (Bio-Rad Laboratories,Inc.,Hercules,CA,USA)with a standard curve based on bovine serum albumin.The primers used for PCR are given in Table S1.

2.6.In vitro assay for ALS activity

ALS activity was assayed as described previously with modifications [10,32].ALS activity was assayed in a 37.5-μL reaction mixture containing 1% (v/v) DMSO,10 mmol L-1MgCl2,100 mmol L-1potassium phosphate buffer (pH 7.0),10 mmol L-1thiamine pyrophosphate,100 mmol L-1sodium pyruvate,0.1 mmol L-1flavin adenine dinucleotide disodium salt hydrate,and 3.75 μg of protein dissolved in 3 μL of elution buffer.The inhibitors used in this study included imazethapyr,imazamox,imazapic,mesosulfuronmethyl,pyribenzoxim,flumetsulam,and flucarbazone-sodium,which were purchased from Toronto Research Chemicals (Ottawa,Canada).

The inhibitors were dissolved in DMSO and added to the reaction mixture at different concentrations,respectively.The mixture was heated at 37°C for 1 h and the reaction was stopped by adding 7.5 μL of 5% H2SO4.Then the mixture was incubated statically at 60°C for 15 min for the decarboxylation of acetolactate to acetoin.The color of acetoin was developed with 31.25 μL of creatine(5 mg mL-1) and 31.25 μL of α-napthol (50 mg mL-1in 4 mol L-1NaOH) at 60 °C for another 15 min.The samples were read by a microtiter plate reader(Varioskan Flash;Thermo Fisher,Waltham,MA,USA)at 540 nm.Data from each line was fit to a nonlinear regression model as described using Sigmaplot 14.0 [33].

3.Results

3.1.Screening for imazethapyr-resistant wheat

To isolate imazethapyr-resistant wheat mutants,we first prepared a common wheat mutagenized seed pool.For this purpose,>200 kg of seeds from spring wheat cultivar ‘Ningchun 4′were mutagenized by EMS.The M1plants were self-bred in the field to produce M2seeds;990 kg of M2seeds were harvested.The M2seeds were treated with 0.05% (v/v) imazethapyr (～1.7 mmol L-1)for 18 h and then placed on a tray in a growth chamber (16 h light/22°C,8 h dark/22°C)(Fig.S1A,B).Plants showing significant growth were visually evaluated after 7–10 days of incubation(Fig.S1C).The putative imazethapyr-resistant mutants were transferred to another growth chamber(16 h light/15°C,8 h dark/15°C)to recover(Fig.S1D).After 2–3 weeks,they were moved to a greenhouse (16 h light/24 °C,8 h dark/19 °C) until harvest (Fig.S1E).In total,36 imazethapyr-resistant plants were obtained from a screen of～ 700,000 M2seeds.

3.2.Cloning of the mutated gene in the imazethapyr-resistant plants

It was previously shown that the mutation of ALS resulted in IMI-resistant plants [12].Therefore,we first attempted to identify the mutated site withinTaALSin our imazethapyr-resistant wheat plants.Similar to most genes in the hexaploid wheat genome,threeTaALShomologs with high similarity are located in the A,B,and D subgenomes;we designated themTaALS-A,TaALS-B,andTaALS-D,respectively (Fig.S2).Two mutant plants (line 1 and line 2) with mutation inTaALS-D,G1880A,were identified in our imazethapyr-resistant plants (Fig.1A,B).The mutation was heterozygous in plant line 1 and homozygous in plant line 2,indicating that the mutation in TaALS that caused imazethapyr resistance was dominant (Fig.1C).Cloning of the mutated genes from additional imazethapyr-resistant plants showed that the mutation ofTaALS-AorTaALS-Balso resulted in imazethapyr resistance in wheat;notably,these genes shared similar mutation sites withTaALS-D(Fig.S3A–D;Table 1).

To further investigate the distribution of mutation sites leading to imazethapyr resistance in wheat and for the preparation of wheat varieties with imazethapyr resistance,we prepared mutagenized seed pools from four other wheat varieties.As shown in Table 1,352 imazethapyr-resistant mutants were isolated from>7,000,000 M2plants(Table 1).Further analysis demonstrated that the mutation of each of three to four sites in each TaALS yielded imazethapyr-resistant wheat plants,and tentaalsalleles producing imazethapyr resistance were isolated(Table 1).Interestingly,moretaals-dplants were identified thantaals-aandtaals-bplants,and the mutants carrying S627N (corresponding to TaALSD) accounted for 78.5% of all imazethapyr-resistanttaalsplants(Table 1).These results show that imazethapyr resistance in wheat can be produced by the mutation of one of four sites in one of three homologous TaALS proteins.taals-dwas the most commonly identified mutant among those mutants carrying a change at the same site,while mutants carrying S627N in TaALS were the most commonly isolated overall,suggesting that the mutation of this site leads to stronger herbicide resistance than other mutations.

3.3.Characterization of imazethapyr resistance in taals plants

Unlike the outcomes in other diploid plants,the mutation of each of the threeTaALSgenes in wheat produced imazethapyr resistance (Table 1).To determine whether there was any difference in imazethapyr resistance among the mutants of the threeTaALShomologs,we usedtaals-a(S630N in TaALS-A),taals-b(S629N in TaALS-B),andtaals-d(S627N in TaALS-D) to generate higher-ordertaalsmutants and to uncover the genetic characteristics of imazethapyr resistance in these differenttaalsmutants.

Phenotypic analysis showed that the growth of the control plants was inhibited significantly following imazethapyr treatment(0.6 mmol L-1imazethapyr is equal to the suggested concentration of imazethapyr used in the field[approximately 75 g ha-1])(Fig.2),indicating that natural wheat is not resistant to imazethapyr,as shown previously[9,10].However,the growth of thetaalsmutants was better than that of the control plants following the same imazethapyr exposure (Fig.2A–C),suggesting that all three homologoustaalsmutants were resistant to imazethapyr.In addition,the growth oftaals-dwas stronger than that oftaals-a,while the growth of thetaals-aplants was stronger than that oftaals-bfollowing treatment with imazethapyr (the difference was obvious at a relatively high concentration of imazethapyr;Fig.2A–C).Thus,taals-dwas more resistant to imazethapyr thantaals-aortaals-b,andtaals-bshowed the weakest resistance among the three singletaalsmutants.This is consistent with the fact thattaals-dwas the most commonly identified among the three mutants(Table 1).Further analysis showed that the losses in fresh weight and dry weight of the threetaalssingle mutants exceeded those of the double mutants,while the losses in the double mutants exceeded those of the triple mutant following imazethapyr treatment (e.g.,0.6 and 2.4 mmol L-1imazethapyr;Fig.2A–C).In summary,the differenttaalsmutants exhibited different degrees of resistance to imazethapyr,and the imazethapyr resistance observed in our wheatTaALSmutants was expressed in a dose-dependent manner.

Table 1 Summary of the identification of imazethapyr-resistant plants and the mutation sites in the taals mutants from five wheat varieties.

3.4.Biochemical basis for the imazethapyr resistance of the taals plants

To understand the mechanism of imazethapyr resistance intaals,an assay for TaALS activity was performed.First,we expressed and purified TaALS proteins from bacteria.As there are three functional copies ofTaALSin the wheat genome,the three TaALS homologs and their corresponding mutant TaALS proteins were expressed in and purified fromEscherichia coli(Fig.S4).

Fig.1.Identification of the mutation sites in imazethapyr-resistant lines 1 and 2.(A) Phenotypic observation of imazethapyr-resistant lines 1 and 2 after imazethapyr screening.The seeds were treated with 1.7 mmol L-1 imazethapyr for 18 h and then placed on a tray in a growth chamber for 6 days.NC4,Ningchun 4 (control plant).Bar=1 cm.(B)G1880A produced the substitution S627N in TaALS-D in lines 1 and 2.(C)Line 1(left)was heterozygous while line 2(right)was homozygous for the G1880A mutation in TaALS-D.An alignment of the TaALS-D open reading frames from lines 1 and 2 was done with ClustalW.The numbers in (B) indicate the positions of the bases.Base number 1880 in TaALS-D is indicated by a red box in (B) and (C).

Our biochemical analysis indicated that the activity of the three homologous TaALS proteins was significantly inhibited by imazethapyr,with a similar I50(i.e.,the inhibitor concentration corresponding to a TaALS activity level midway between the upper and lower asymptotes) (Fig.3;Table S2).Meanwhile,the enzymatic activities of the mutated TaALS proteins,including m-TaALS-A(S630N),m-TaALS-B (S629N),and m-TaALS-D (S627N),were insensitive to inhibition by imazethapyr (an IMI;Fig.3).

As each of the three or four mutations in TaALS caused imazethapyr resistance,we used TaALS-D as an example to test the effect of imazethapyr on the activity of each of mutated TaALS protein.Again,the activity of TaALS-D was inhibited by imazethapyr;however,the mutated TaALS-D proteins,including m-TaALS-D(A96T),m-TaALS-D (A179V),and m-TaALS-D (S627N),were not obviously inhibited by imazethapyr.Further,the activity of m-TaALS-D (P171L) was decreased and obviously slower than that of wild-type TaALS-D as the concentration of imazethapyr increased(Fig.4;Table S3).This is consistent with the observation that only 1tasls-d (C512T)plant was identified among 352 imazethapyr-resistant plants (Table 1).These data suggest that the different TaALS mutant proteins exhibited different degrees of tolerance to inhibition by imazethapyr.

Further analysis indicated that the trends in inhibition of fresh weight and dry weight of the aboveground parts in control andtaals-d (G1880A)plants by imazethapyr were generally consistent with the decreases in TaALS-D and m-TaALS-D(S627N)activity following imazethapyr exposure (Fig.5;Table 2).These results demonstrate that the mutation ofTaALSincreased the tolerance of TaALS to imazethapyr inhibition and caused the imazethapyr resistance observed in ourtaalsmutants.

Table 2 The effects of different ALS inhibitors on TaALS-D and m-TaALS (S627N) activity based on the I50 and on the growth of control and taals-d plants based on the GR50.

3.5.Resistance of the taals plants to different IMI herbicides

Fig.2.The effect of imazethapyr on taals growth after 4 days of imazethapyr treatment.(A) Phenotypic observation of taals mutants treated with imazethapyr at different concentrations.Bar=3 cm.(B,C)The relative fresh weights(B)and relative dry weights(C)of the aboveground parts of the plants analyzed.The mean fresh weights and dry weights (% of control) were calculated from three independent biological replicates.Error bars indicate the standard deviation (SD). n=30.

Fig.3.The effect of imazethapyr on the activity of wild-type and mutated versions of TaALS.The data represent the mean (% of control) at each concentration of imazethapyr.Lines represent the fitted line of the mean from three independent biological replicates with two technical replicates for each data point.Error bars indicate the SD.

Imazethapyr is one of six types of IMI herbicides.These herbicides are characterized chemically by three distinct moieties (the imidazolinone ring,carboxylic acid,and aromatic backbone ring),and each of these moieties is required for herbicide activity[18].To determine whether ourtaalsplants could tolerate other IMI herbicides,we selected two additional IMI herbicides (imazamox and imazapic) and the mutanttaals-d.

Fig.4.The effect of imazethapyr on the activity of wild-type and mutated versions of TaALS-D.The data represent the mean (% of control) at each concentration of imazethapyr.Lines represent the fitted line of the mean from three independent biological replicates with two technical replicates for each data point.Error bars indicate the SD.

Phenotypic analysis revealed that the growth of the control plants was obviously inhibited by imazamox or imazapic,whereas the growth oftaals-d (G1880A)was not obviously affected following the same treatment,similar to our results using imazethapyr(Fig.6).Thus,taals-dwas resistant to different IMI reagents to a similar degree.This suggests that the resistance mechanism may be the same.

Fig.5.The effect of imazethapyr on the activity of TaALS-D and m-TaALS (S627N) and on the growth of control and taals-d plants.(A) Phenotypic observation of taals-d(G1880A) treated with imazethapyr.(B) The data represent the mean (% of control) of the plant aboveground weight or activity of TaALS and m-TaALS (S627N) after imazethapyr treatment(indicated by the top x axis and bottom x axis,respectively).Lines represent the fitted line of the mean from three independent biological replicates.Error bars indicate the SD. n=30 for the growth analysis.

Biochemical analyses using purified TaALS-D showed that the activity of the enzyme was obviously inhibited by different IMI reagents with a similar I50,and that the tolerance exhibited by m-TaALS-D(S627N)to different IMI reagents was similar to its tolerance to imazethapyr(Fig.7;Table S4).These results indicate that the different IMI herbicides had similar effects on the activity of TaALS and that thetaalsplants were tolerant to different types of IMI herbicides.

3.6.The effect of a non-IMI ALS inhibitor on taals plant growth

Besides IMIs,four other types of chemicals,sulfonylureas(SUs),triazolopyrimidines (TPs),pyrimidyloxybenzoates (PTBs),and sulfonlyaminocarbonyl-triazolinones (SCTs),can inhibit ALS activity in plants [34].We used m-TaALS-D (S627N) andtaals-d(G1880A)to determine whether the above-mentioned mutation in TaALS could also affect the inhibition of ALS activity andtaalsgrowth by these non-IMI inhibitors.

Fig.7.The effect of IMI herbicides on the activity of wild-type and mutated TaALSD.The data represent the mean (% of control) at each concentration of IMI.Lines represent the fitted line of the mean from three independent biological replicates with two technical replicates for each data point.Error bars indicate the SD.

All four chemicals mentioned above inhibited TaALS-D activity(Fig.S5).The sensitivity of m-TaALS-D (S627N) to flumetsulam (a representative TP) and pyribenzoxim (a representative PTB) was similar to that of TaALS-D;in contrast,its sensitivity to mesosulfuron-methyl (a representative SU) and flucarbazonesodium (a representative SCT) was slightly lower than that of TaALS-D (Fig.S5).Consistent with this observation,there was no obvious difference in growth between the control andtaals-dplants following treatment with flumetsulam and pyribenzoxim,andtaals-dexhibited slightly more tolerance than the control plants to flumetsulam and flucarbazone-sodium (Figs.S5,S6).In addition,the control andtaals-dplants were sensitive to flumetsulam,mesosulfuron-methyl,and flucarbazone-sodium,but tolerant to pyribenzoxim (Figs.S5,S6).These results demonstrate that the mutation of ALS on the above-mentioned sites did not produce obvious tolerance to non-IMI ALS inhibitors,and that wheat tolerates pyribenzoxim through an unknown,ALS-independent pathway.

4.Discussion

Weeds have strong environmental adaptability,which enables them to possess more growth factors than crops in the field [35].Therefore,field crop production can be reduced by a large number of weeds [36–38].Compared with physical or biological weed management,weed control with herbicides is energy-saving and requires less labor.Thus,the breeding of herbicide-resistant wheat varieties can help control weeds in wheat fields more economically and effectively,and this in turn can improve wheat yields and quality.

In the present study,we found that the mutation of each of three to four sites in one of threeTaALSgenes conferred various degrees of IMI resistance to wheat plants (Figs.1 and 2;Table 1).The observed herbicide resistance of our wheattaalsmutants was a dominant trait and exhibited dose-dependent expression(Figs.1B,2).These results are important for the cultivation of new wheat varieties with increased IMI herbicide resistance,which may minimize crop damage caused by inaccurate control of the herbicide concentration during application.This work also provides a foundation for the breeding and cultivation of wheat varieties with herbicide resistance.

Our biochemical analysis showed that IMI herbicides kill common wheat by inhibiting the enzymatic activity of TaALS (Figs.3 and 4).The mutation of TaALS resulted in reduced inhibition by IMIs;thus,our wheattaalsmutants were resistant to IMI herbicides(Figs.4–7).Interestingly,the growth responses of our control andtaals-dplants to ALS inhibitors were consistent with the effects of these inhibitors on the enzymatic activities of TaALS-D and m-TaALS-D (S627N) (Figs.5 and 7).However,both TaALS-D and m-TaALS-D (S627N) were sensitive to non-IMI ALS inhibitors(Fig.S6),and the growth of the control andtaals-dplants was repressed by these same inhibitors (except for pyribenzoxim,to which wheat is resistant by an unknown,ALS-independent pathway;Figs.S5 and S6).These results suggest thatalsplants are tolerant only to IMI ALS inhibitors (herbicides),and they imply that protein modeling andin vitroenzymatic assays may identify candidates with herbicidal activity for preliminary screening during new herbicide design.

Genetic transformation techniques have been used to successfully breed herbicide-resistant crops [24,39,40].Recently,gene editing technology has been applied to herbicide-resistant crop breeding [41–45].Genetic screening of a mutant pool constructed by chemical mutagenesis is another means of crop breeding,and it has several advantages[46–48].First,it is a transgene-free method so it is not restricted by concerns about transgenic safety.Second,transgenic method depend on understanding the target gene to be transformed[49],while revealing the target gene and the mutation(s) that leads to desired resistance will help to create resistant plant by gene editing.It was observed that the exact mutation that confers the highest level of herbicide resistance can vary among different crops.For example,the mutation of TaALS-D at S627N may yielded the greatest resistance to IMI in wheat (Fig.2),while W560L in feral radish and W563C/W563S in cotton yielded the highest levels of IMI resistance[27,50].Therefore,genetic analysis of the mutants will help to generate herbicide-resistant crops by transgenic or gene editing method.Finally,the technical requirement is relatively simple for resistant crop line screening from a mutagenized pool,while the isolation frequency of resistant lines is relatively high.For instance,there are three copies of TaALS in the wheat genome,and the IMI resistance trait was dominant(Figs.1B,S2);thus,one resistant wheat plant was isolated on average from about every 20,000 M2seeds under our conditions(Table 1).In addition,genetic screening of mutagenized seed pools can be used to obtain desired agricultural traits that are mediated by unknown genes.The mutations induced by EMS will be accompanied with undesired and uncharacterized mutations in the whole genome;those mutations need to be eliminated by backcrossing.Therefore,we believe that genetic screening using a mutagenized pool is an effective and important means of breeding herbicide-resistant crops with additional desired agricultural traits.

CRediT authorship contribution statement

Ligeng Ma and Xing Wang Deng:supervised the project and acquired the funding;Zhuo Chen,Zheng Wang,Yanfang Heng,Jian Li,Jiawei Pei,and Ying Cao:conducted the experiments and analyzed the data;Ligeng Ma and Zhuo Chen:wrote draft the manuscript;Ligeng Ma,Zheng Wang,Zhuo Chen,and XingWang Deng:edited and finalized the manuscript.All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that there is no conflict of interest.

Acknowledgments

We thank Dr.Jessica Habashi for critical reading of the manuscript.We thank Drs.Xiaoxue Wang and Kuanji Zhou for their help for the preparation of mutant pools at the very beginning stage of the screening.This work was financially supported by the National Key Research and Development Program of China(2017YFD0101001),Beijing Municipal Government Science Foundation (IDHT20170513),and Peking University Institute of Advanced Agricultural Sciences.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.11.001.