Development of herbicide resistance genes and their application in rice

Man Jin, Lei Chen, Xing Wang Deng, Xiaoyan Tang

a Shenzhen Institute of Molecular Crop Design, Shenzhen 518107, Guangdong, China

b Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, Guangdong, China

c Institute of Plant and Food Sciences, Department of Biology, Southern University of Science and Technology, Shenzhen 518055, Guangdong, China

d Shenzhen Agricultural Technology Promotion Center, Shenzhen 518055, Guangdong, China

Keywords:Rice Herbicides Herbicide resistant genes Gene editing Mutant

ABSTRACT Rice is one of the most important food crops in the world.Weeds seriously affect the rice yield and grain quality.In recent years, there are tremendous progresses in the research and application of herbicideresistant genes in rice worldwide.This article reviews the working mechanisms of six herbicides(glyphosate,glufosinate, acetolactate synthase inhibitor herbicides, acetyl-CoA carboxylase inhibitor herbicides,hydroxyhenylpyruvate dioxygenase(HPPD)inhibitor herbicides and dinitroaniline herbicides),the resistance mutations of the corresponding herbicide-target genes, and the herbicide detoxification mechanisms by non-target genes.Examples are provided on herbicide-resistant rice materials obtained by transformation of exogenous resistance genes, by artificial mutagenesis and mutant screening, and by modifying the target genes through gene editing.This paper also introduces the current application of herbicide-resistant rice,points out problems that may be caused by utilization of herbicide resistant rice and solutions to the problems, and discusses the future prospects for the development of herbicideresistant rice.

1.Introduction

Rice is the staple food for more than half of the world’s population.It is one of the most important food crops in the world.Rice production occupies a very important position in agriculture, particularly in Asian countries.Weed is an important factor affecting rice production [1].Weeds compete with rice for nutrients, water,sunlight, and space.The presence of weeds also increases the occurrence of diseases and insect pests.These problems lead to varying degrees of reduction in rice yield and grain quality.It was reported that the rice yield can be reduced by more than 40% due to weed damages [2].Rice is the major crop in China.As urbanization in China speeds up, a large number of laborers are flowing to cities, and the problem of labor shortages in ricegrowing areas is increasing.This has gradually changed rice cultivation from transplanting to direct seeding, which often results in more frequent and serious weed damages in China.Traditional measures to control weeds in rice fields include combination of tillage,irrigation,and hand-weeding,which require extensive labors and other resources.Rational utilization of herbicides is the most effective way in weed control.However, most of the herbicides that kill weeds in rice field also cause certain damages to the rice.Particularly,there is no chemical that can effectively control weedy rice,a notorious and widely-occurring weed that is very similar to cultivated rice in taxonomy and physiology,without damaging the cultivated rice[2,3].The problem of weeds(especially weedy rice)in rice production can be effectively solved by the application of herbicide-resistant rice in combination with corresponding herbicides.Therefore, herbicide-resistant rice has received more and more attentions and has become an important direction of genetic breeding.In recent years, researchers have conducted extensive studies on herbicide-tolerant plants and the resistance mechanisms.A series of herbicide-resistant rice materials were obtained through artificial mutagenesis and mutant screening, targeted mutagenesis of the herbicide-resistant genes, or introducing exogenous resistance genes into rice through transformation.This article provides an overview of a few major herbicides and their working mechanisms, the resistant genes for these herbicides,and the commercial application of herbicide-resistant rice in agriculture production.Future development and prospects of herbicide-resistant rice are also discussed.

2.A few major herbicides and their corresponding target genes

2.1.Glyphosate and EPSPS

Fig.1.Working mechanisms of the herbicides.(A)Glyphosate mode of action(Adapted from Dill[5]with the permission of John Wiley&Sons,Inc.).(B)Glufosinate mode of action.GS,glutamine synthetase;GOGAT,glutamate synthase;(C)ALS inhibitor herbicide mode of action.ALS,acetolactate synthase.(D)ACCase inhibitor herbicide mode of action.ACCase,acetyl-CoA carboxylase.(E)HPPD inhibitor herbicide mode of action.HPPD,hydroxyphenypyruvate dixygenase.The red x in the figure means inhibition of the reaction.

Glyphosate is a phosphate compound with a stable C-P bond and targets the 5-enolpyruvoylshikimate-3-phosphate synthase(EPSPS) in plant [4].EPSPS is a chloroplast enzyme catalyzing the production of 5-enolpyruvylshikimate-3-phosphate (EPSP) from phosphoenolpyruvate(PEP)and shikimate-3-phosphate(S3P).This is a key step for the plant to synthesize tryptophan, tyrosine and phenylalanine,as well as hormones,flavonoids,lignin,ubiquinone,and other phenolic compounds.Glyphosate forms a stable EPSPSS3P-glyphosate complex with EPSPS in plants by competing with PEP, which disrupts the EPSPS activity.As a result, the synthesis of aromatic amino acids necessary for protein biosynthesis is obstructed, shikimate is excessively accumulated, and the synthesis of hormones and secondary metabolites required for plant growth is also inhibited.These in turn cause disorders in plant growth and metabolisms, resulting in plant death [5] (Fig.1A).

At present,50 different species of weeds have been found in the world with various levels of resistance to glyphosate,among which 15 species of weeds are resistant to glyphosate due to the mutations in EPSPS [6].The mutation sites are Thr102to Ile/Ser, Ala103to Val, and Pro106to Ser/Ala/Leu/Thr.For amino acids mutated at Thr102and Pro106,conversions of Thr102to Ile and Pro106to Ser give rise to the highest level of resistance [6].

2.2.Glufosinate and glutamine synthetase

Glufosinate (phosphinothricin, PPT) is a non-selective broadspectrum herbicide targeting at glutamine synthetase(GS).GS converts glutamate and ammonia into glutamine,and is a key enzyme necessary for plant nitrogen metabolism.As an analog of glutamate, PPT competes with the natural substrate of GS, which inhibits the nitrogen assimilation and causes excessive accumulation of ammonia in plants, thereby causes the reduction of photosynthetic activity, the destruction of chloroplast structure, the degradation of glyoxylic acid, and eventually the plant death [7](Fig.1B).

The occurrence of glufosinate-resistant weeds is relatively slow.So far, only one goose grass and three ryegrass have been found with certain level of resistance to glufosinate [8].Research on the resistant Italian ryegrass shows that, upon Asp171replaced by Asn, the change in amino acid polarity may be one of the reasons for the decreased sensitivity of GS to glufosinate [9].Studies of the alfalfa GS show that the protein exhibits certain level of glufosinate resistance when the amino acid at position 207 is Gly, position 245 is any amino acid but Gly, and position 332 is Arg or Lys [10].The rice glutamine synthetase mutant (OsGS1; 1), with Gly at position 59 and Arg at position 296,has enhanced tolerance to glufosinate [11].

2.3.Acetolactate synthase (ALS) inhibitor herbicides and ALS

ALS inhibitor herbicides kill weeds by inhibiting ALS.ALS is a key enzyme in the synthesis of branched-chain amino acids in plants, such as valine, leucine and isoleucine [12].Herbicides in this group inhibit the activity of ALS and prevent the synthesis of branched-chain amino acids in the plant, which in turn interfere the protein synthesis, cell division and plant growth, and eventually lead to plant death (Fig.1C).Currently, the widely used ALS inhibitor herbicides include imidazolinones (IMI), sulfonylureas(SU), triazolopyrimidines (TP), pyrimidinylthiobenzoates (PTB),and sulfonylamino-carbonyl-triazolinones (SCT) [12].

Studies on resistant weeds have shown that, although some weeds develop resistance by improving the herbicide detoxification ability, in most cases, mutations in the target ALS genes are the main reason for herbicide resistance.At present, a total of 66 different weed species have developed resistance to ALS inhibitor herbicides due to mutations in ALS,including 29 amino acid mutations in the following 8 positions:Ala122(5), Pro197(11), Ala205(2),Asp376(1), Arg377(1), Trp574(4), Ser653(3), and Gly654(2).Among them,Pro197can be substituted by 11 other amino acids,includingThr, Ala, Arg, Asn, Gln, His, Ile, Leu, Ser, Thr and Tyr [13].Amino acid mutations in ALS can change the enzyme structure or spatial conformation,reducing the protein affinity for herbicides and thus developing resistance.Different mutation sites and the types of substituted amino acids determine the level of resistance and the range of resistance to ALS inhibitor herbicides (Table 1).

Table 1 Some ALS mutations and their resistance to herbicide.

Table 2 Some ACCase mutations and their resistance to herbicide.

2.4.Acetyl-CoA carboxylase (ACCase) inhibitor herbicides and ACCase

ACCase catalyzes the carboxylation of acetyl-CoA in organisms to produce malonyl-CoA, which provides a substrate for the synthesis of fatty acids and many secondary metabolites.It is therate-limiting enzyme for fatty acid synthesis[23].ACCase inhibitor herbicides disrupt fatty acid synthesis by inhibiting ACCase,which in turn hinder the production of lipid components in the membrane system, causing cell structure damage and ultimately the plant death (Fig.1D).

ACCas inhibitor herbicides include the following three categories:aryloxyphenoxypropionates (APPs), such as quizalofop-p-ethyl, diclofop-methyl, and oxazole metamifop, etc.;cyclohexanediones (CHDs), such as sethoxydim, clethodim, and tralkoxydim, etc.; and phenylpyrazoline (DEN), such as pinoxaden[24].The carboxyl transferase(CT)domain of the plastid ACCase is the target region for these herbicides [25].Research on resistant weeds found that mutations of Ile1781Leu, Trp1999Ser/Cys, Trp2027-Cys, Ile2041Asn, Asp2078Gly, Cys2088Arg, and Gly2096Ala all exist in the CT domain.These mutations confer resistance to different ACCase inhibitor herbicides in plants(Table 2).Among these mutations, Ile1781Leu mutation is the most widespread in plants [26].

Table 3 Herbicide-resistant rice obtained through gene editing.

2.5.Hydroxyphenypyruvate dixygenase (HPPD) inhibtor herbicides and HPPD

HPPD exists widely in a variety of organisms, catalyzing the conversion of hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA), the precursor for the biosynthesis of plastoquinone and tocopherol in plant [40] (Fig.1E).HPPD inhibitor herbicides chelate with the Fe2+in the enzyme active site, which blocks the binding of HPPD with substrate HPPA,resulting in competitive inhibition of HPPD.Plastoquinone and tocopherol are precursors for carotenoid biosynthesis.Carotenoid plays critical roles in photosynthesis.Inhibition of HPPD blocks the photosynthetic electron transfer, causing plant bleaching and death [41].

At present,Amaranthus palmeriis the main weed that has developed resistance to HPPD inhibitor herbicides.Researches show that the sequences of HPPD in the resistant and sensitiveAmaranthusare identical.However,the expression level of HPPD was increased in the resistantAmaranthus,which may be accountable for the elevated resistance to HPPD inhibitor herbicides[42,43].Maeda et al.[44]recently identified a rice gene,HIS1(HPPD inhibitor sensitive 1),that confers resistance to benzobicyclon (BBC) and other β-triketone herbicides.HIS1encodes an Fe2+/2-oxoglutarate-dependent oxygenase that detoxifies β-triketone herbicides by catalyzing their hydroxylation.Rice varieties sensitive to the HPPD inhibitor herbicides carry a 28-bp deletion of theHIS1coding region.

2.6.Dinitroaniline herbicides and tubulin genes

Dinitroaniline herbicides were extensively used in weeding of soybeans and cottons.It can control not only annual grass weeds,but also some annual broad-leaved weeds.Dinitroaniline herbicides including trifluralin, pendimethalin and ethalfluralin can bind to tubulin and interrupt the polymerisation of microtubules,arresting cell division and elongation and resulting in plant death[45].Molecular structural modeling and analysis of tublin resistance mutation in plants have demonstrated that dinitroaniline most likely interacts with α-tubulin to disrupt microtubule polymerisation.

A few weeds, such asEleusine indica,Setaria viridis,Alopecurus aequalis, andLolium rigidumhave developed resistance to dinitroaniline.The resistance was due to mutations in α-tubulin.α-Tubulin mutations of Thr239Ile and Met268Thr were found in theE.indicaresistant type [46,47], and Leu136Phe and Thr239Ile in the resistantS.Viridis[48].In addition,Val202Phe,Leu125Met,Leu136Phe mutations were identified in the resistantA.Aequalis[49].Val202-Phe, Thr239Ile and Arg243Met/Lys mutations were present in the resistantL.rigidum[45,50,51].

3.The working mechanisms of non-target herbicide-resistant genes

3.1.Glyphosate degrading enzymes and glyphosate-Nacetyltransferase

Studies have shown that microorganisms in the soil, including bacteria, actinomycetes, and fungi, can degrade glyphosate.From these microorganisms, researchers have isolated a series of genes that confer resistance to glyphosate.For example, igrA (increased glyphosate resistance A) can break the C-P bond of glyphosate to generate sarcosine and inorganic phosphorus[52].Glyphosate oxidoreductase (GOX) can break the C-N bond of glyphosate to form aminomethyl phosphate (AMPA) and glyoxylate [53].In addition,glycine oxidase (GO) and D-amino acid oxidase (DAAO) also play a role in the degradation and detoxification of glyphosate[53].Glyphosate N-acetyltransferase (GAT) transfers the carboxyl group from CoA to the N-terminus of glyphosate to generate N-acetyl glyphosate, which is a product without herbicide toxicity [54].

3.2.N-acetyltransferase

The N-acetyltransferase (phosphinothricin acetyltransferase,PAT)can acetylate the toxic L-PPT,thereby detoxifying glufosinate.Balthazor et al.[55] has cloned the bialaphos resistant gene (bar)fromStreptomyces hygroscopicusand phosphinothricin acetyltransferase gene(pat)fromStreptomyces viridochromo,respectively,that confer glufosinate resistance.With the development of microbial genome sequencing,the nucleotide sequences encoding PAT family proteins have been predicted in many microorganisms,but none of them have been confirmed with glufosinate resistance or utilized in commercialized genetically modified crops.Currently, onlybarandpatare used for commercialization [56].

3.3.Cytochrome P450s

Cytochrome P450 super family not only participate in the metabolic processes of hormones,lipids,and secondary metabolites,but also play an important role in the detoxification of various herbicides by hydroxylation or dealkylation of ACCase-, ALS-, and photosystem II (PS II)-inhibitors [57].However, up to date, only one cytochrome P450 gene has been identified in rice that can enhance the plant tolerance to Bentazone, a selective herbicide that can inhibit photosynthesis.Disruption of this gene makes the plant sensitive to Bentazone[57].Further exploration of this super gene family for enhanced capability to detoxify various herbicides will be beneficial to molecular manipulation and breeding of herbicide resistant crops.

4.Application of herbicide resistance genes in rice

4.1.Overexpression of foreign genes in rice

The EPSPSs of plant origin are generally sensitive to glyphosate inhibition,but the EPSPS isolated from glyphosate-resistant bacteria and their optimized forms have a reduced binding affinity to glyphosate and thus are more tolerant to glyphosate.Most of the glyphosate-resistant rice lines are generated via transgenic approach by introducing EPSPS genes from resistant bacteria or their optimized forms into the plant.For example, Zhao et al.[58] transferred G6 EPSPS derived fromPseudomonas putidainto rice cultivar Xiushui 110 to obtain a transgenic line resistant to glyphosate.Chhapekar et al.[59]transformed a codon-optimized CP4EPSPSgene derived fromAgrobacterium tumefaciensinto rice variety IR64 and obtained transgenic plants that could tolerate 10xthe commercial recommended dose of glyphosate.Cui et al.[60]and Yi et al.[61] cloned the glyphosate resistance genesaroAJ.sp(encoding EPSPS) andI.variabilis-EPSPSfromJanibactersp.andIsoptericola variabilis, respectively, and transformed them into thejaponicarice variety Zhonghua 11 and theindicarestorer line Minghui 86,and obtained transgenic lines of high resistance to glyphosate.There are also a few transgenic events that combined the resistantEPSPSgenes with the non-target glyphosate-resistant genes to enhance the rice resistance to glyphosate.For example,Fartyal et al.[62] transferred the mutant riceEPSPSgene and theigrAgene intoindica Swarna, and obtained transgenic rice with high glyphosate-resistance and low herbicide residue.

Glufosinate-resistant rice lines were generated mainly by transformation ofbarandpatgenes to enhance the resistance to glufosinate.As early as 1996, Oard et al.[63] used gene gun to transfer thebargene into Gulfmont, IR72, and Koshihikari varieties; they further conducted field experiments and obtained transgenic glyphosinate-resistant rice varieties.A few glufosinate-resistant transgenic rice varieties were also generated in China.For example,thebargene was transferred into two-line or three-line restorer rice lines such as Xiushui 04, Minghui 63, R187, D68, and E32[64,65], as well as upland rice lines 297 and 502 [66].Cui et al.[56] transferred theRePATgene isolated from aRhodococcussp.strain into Zhonghua 11 rice.The T0transgenic lines showed strong resistance to glufosinate,and the T2generation showed no significant difference from the control plants in the agronomic traits.

Cytochrome P450 is widely present in organisms.Overexpression of cytochrome P450 in rice can enhance the resistance to herbicides, generally with different modes of actions [67].Kawahigashi et al.[68] transformed the human cytochrome P450 genesCYP1A1,CYP2B6andCYP2C19collectively in thejaponicarice Nipponbare,and the resulting transgenic plants showed enhanced resistance to different types of herbicides,including the root elongation inhibitors (pyributicarb), very long-chain fatty acid inhibitors (acetochlor, metolachlor, and thenylchlor), photosynthesis inhibitors (chlortoluron), and carotenoid biosynthesis inhibitors(norflurazon).The human cytochrome P450 genes CYP2C9 and CYP2C19 were also individually overexpressed in rice; the resulting CYP2C9 transgenic line 2C9-57 displayed resistance to the sulfonylurea herbicide chlorsulfuron,and the CYP2C19 transgenic line 2C19-12 was tolerant to mefenacet, metolachlor, norflurazon, and pyributicarb [69].

4.2.Rice gene editing

In recent years,gene editing technology,especially CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR associated 9) mediated genome editing has been widely used in the genetic modification of microorganisms, animals and plants.The CRISPR/Cas9 technology recruits Cas9 protein to the target genomic DNA under the guidance of sgRNA and generates DNA double strand break (DSB) at the target site, inducing nonhomologous end joining (NHEJ) and homologous recombination(HR) pathways in the cell, via which to achieve precise genome modifications such as targeted knockout, replacement, and insertion of DNA fragment [70].

Table 3 summarizes the gene editing tools deployed in generating herbicide-resistance in rice and the obtained mutant nucleotides and resistant loci.Li et al.[71] used the CRISPR/Cas9-mediated NHEJ pathway to perform site-directed replacement of the Thr102Ile and Pro106Ser bases of the riceEPSPSgene and sitedirected replacement of the riceEPSPSgene fragment with the same DNA fragment containing Thr102Ile and Pro106Ser (TIPS)mutations.They obtained rice materials resistant to glyphosate,with the substitution sites and the insertion mutation sites stably passed on to the next generation.While the heterozygous TIPS mutant plant grew normally, plant carrying homozygous TIPS mutation could not survive.This is likely because that the TIPS mutation confers higher tolerance to glyphosate but also reduces the EPSPS enzymatic activity.Nonetheless, the TIPS mutant is still a valuable gene for developing glyphosate resistant crops.Recently, it was reported that overexpression of theTIPS-OsEPSPSgene resulted in field level glyphosate tolerance and higher grain yield in rice [72].Besides the TIPS mutant, Sun et al.[73] used the CRISPR/Cas9-mediated HR pathway to introduce two discrete mutation sites, Trp548Leu and Ser627Ile, on riceALSgene, and obtained mutant materials with resistance to bispyribac sodium.

With the development of base editing system, the CRISPR/Cas9 technology has changed from a ‘‘scissors” that can cut the double strand DNA to a‘‘corrector”that can modify specific bases,opening the door to precise genome editing[74].At present, the base editing system can be divided into cytosine base editors(CBE)that can change C to T or G to A, and adenine base editors (ABE) that can change A to G or T to C,according to the different base modification enzymes that are fused to the system.Shimatani et al.[75] used Target-AID (a cytosine editor) to perform site-directed editing of the riceALSgene, mutating the 287th base from C to T.The resulted rice callus with Ala96Val mutation showed resistance to the IMI herbicides.Zhang et al.[76] used a cytosine base editor to create a series of missense mutations in the Pro171and/or Gly628codons ofALSgene to confer herbicide tolerance in rice.Precise base editing of the a-tublin homologue geneOsTubA2was carried out using adenine base editor rBE14, converting T to C,resulting in the Met268Thr replacement.This mutation confers resistance to dinitroaniline herbicides in rice and is stably inherited in the subsequent generations [77].

By constructing the sgRNA library and combining the use of cytosine and adenine base editors,the target gene can be randomly edited to obtain various types of base mutations.Kuang et al.[78]proposed a base-editing mediated gene evolution (BEMGE)method,which uses cytosine and adenine base editors and a sgRNA library that covers the entire length of the target gene coding region, to effectively induce various mutations in the target gene.Using the BEMGE method to edit the riceALSgene, a series of rice mutants, including Pro171Phe, Pro171Leu, Pro171Ser, Arg190His,Ala152Thr, and Ala154Thr, were obtained.The Pro171Phe mutant was the most resistant to bispyribac sodium.Liu et al.[79]designed sgRNAs based on the ACCase CT domain sequence and constructed three mutant libraries eABE, eBE3, and eCDA.Ile1879-Val, Trp2125Ser, and Cys2186Arg mutants showing resistance to haloxyfop-R-methyl were found in the eABE and eBE3 libraries.Li et al.[80] combined the cytosine editor A3A-PBE and adenine base editor PABE-7 to construct a STEMEs system (saturated targeted endogenous mutagenesis editors).Using STEMEs to edit the riceACCasegene, mutants with the Ser1866Phe, Ala1884Pro,Pro1927Phe, and Trp2125Cys substitutions were obtained.Among them, the resistance of Pro1927Phe and Trp2125Cys mutants to herbicide haloxyfop is sufficiently high that can be used for weed control in field.

4.3.Herbicide-resistant rice obtained by artificial mutagenesis and mutant screen

Artificial mutagenesis refers to the use of physical factors, such as X-rays, gamma rays, ultraviolet rays, lasers, etc., or chemical mutagens, such as sodium azide (AZ), ethyl methane sulfonate(EMS), and methyl-nitrosourea (MNU), etc.to induce mutations in the plant genes, followed by screening of the mutant plants of desired phenotype.

The Louisiana State University Agricultural Center used EMS to treat rice variety AS5310,then screened the mutant library by imazethapyr spray, and obtained a resistant mutant with Gly628Glu mutation of theALSgene.Using this mutant, they bred resistant varieties CL121 and CL141 through crossbreeding.Subsequently,the center screened an EMS mutant library derived from theindicarice variety Cypress and obtained the Ser627Asn mutant ofALSgene.Ser627Asn mutant was used to breed the CL161 rice variety and many other Clearfield rice varieties that are resistant to imidazolinone herbicides [81].Then Argentine scholars carried out EMS mutagenesis on the local variety IRGA417 and obtained the Ala96-Thr mutant of ALS.This mutant was used to breed Puita’Intacl[82].Shenzhen Xingwang Biological Seed Industry Co.,Ltd.in China conducted a large scale EMS mutagenesis on an eliteindicacultivar Huanghuazhan(HHZ).They screened the mutant library by spraying the seedlings or soaking the seeds with imazapyr and obtained three more mutants of theALSgene (Ala96Val, Trp548Met, and Trp548Cys).The Trp548Met mutant converted TG at bases 1642 and 1643 to AT of theALScoding region, leading to the mutation of Trp548(TGG)to Met(ATG)[83,84].Compared with the Ser627Asn mutant that is resistant mainly to the IMI herbicides,the Trp548Met mutant displayed higher levels of resistance to all five families of ALS-inhibitor herbicides.Trp548Met was used to breed the Jietian rice varieties [84].Jietian is a Chinese name meaning field free of weeds.Field studies indicated that the Trp548Met mutation had no detectable effect on the growth and yield of rice [84].

Agriculture Victoria Services Pty Ltd used sodium azide(AZ)and methyl-nitrosourea (MNU) to mutagenize rice seeds and obtained a mutant resistant to quizalofop-ethyl.This mutant has a G to A mutation in the carboxyl transferase coding region ofACCase,resulting in the Gly2096Ser mutation and resistance to ACCase inhibitor herbicides [85].The first approved Provisia rice PVL01 is derived from BASF 1-5.BASF 1-5 is an unpublished rice line resistant to ACCase-inhibitor herbicides and containing Ile1781Leu mutation in the ACCase protein [86].

The herbicide resistant rice materials obtained through artificial mutagenesis set a foundation for breeding of commercial rice varieties such as Clearfield rice and Jietian rice to ALS inhibitor herbicides and Provisia rice resistant to ACCase inhibitor herbicides.

5.Application of herbicide-resistant rice in commercial production

Herbicide-resistant rice varieties that have been commercialized included Liberty Link, Provisia, Clearfield rice, and Jietian varieties.

Liberty Link rice confers resistance to glufosinate due to the introduction ofpatgene fromStreptomyces hygroscopicus.Two varieties, LLRICE06 and LLRICE62, were approved in the United States in 1999 to be released into the environment[87].Later they were approved for food or feed in the United States in 2000 and then in Canada in 2006[88].Subsequently,LLRICE62 was approved for consumption in Mexico, Russia, Australia, New Zealand, and Honduras.However, in 2006, the unapproved LLRICE601 rice flowed into the European market, which had many impacts on rice trade, prices, and rice producers and consumers.This event led to the tightening of the EU’s supervision on genetically modified rice[87,88].

Provisia rice resistant to ACCase inhibitors was generated by BASF in Germany through non-transgenic approach.PVL01 is the first approved Provisia rice.It was approved for release by the Louisiana Agricultural Experiment Station in 2017 and was expected to enter the US market in 2018[89].The Provisia system can complement the Clearfield system,and growers can alternately use ALS-inhibitor herbicides and ACCase inhibitor herbicides for weed control [90].

Clearfield rice carrying the Ser627Asn mutation in the endogenousALSgene is currently the most prominent herbicide resistant rice in cultivation[91].Clearfield rice confers resistance to IMI herbicides, a class of highly efficient herbicides effective to a broad spectrum of weeds.It was first commercialized in the United States in 2002.Clearfield rice is currently planted in many countries around the world, mainly in the United States, Brazil, Uruguay,Argentina, and other countries in South America.There are 10 countries in Europe growing rice, with a total area of~ 600,000 ha.In 2012, Italy planted about 235,000 ha of rice, and more than 60,000 ha were Clearfield rice, accounting for ~25% of the country’s total rice planting area.By 2014, Clearfield rice has constituted ~ 60% of rice acreage in Arkansas, where most of the U.S.rice is grown [2].Many Clearfield rice varieties have been released along with some negative consequences [2].First, the residual activity of IMI herbicides could injure the rotation crops.In many Asian countries,rice field is planted 2-3 times a year with different crops.Because the IMI herbicides have a long turnover time that may cause problems to subsequent cropping, Clearfield rice has not be cultivated in Asian countries other than Vietnam and Malaysia.Second,due to the close genetics between cultivated rice and weedy rice, resistant gene could transfer from Clearfield rice to weedy rice through natural hybridization.Third, resistant weeds to IMI herbicides also emanated from spontaneous mutation in theALSgene.Thus, implementation of this technology requires long-term planning, appropriate stewardship, institutional collaboration and oversight to keep the negative impacts in check [2,91].

Jietian rice varieties carry the Trp548Met mutation ofALSgene.This mutation was obtained in 2011 from EMS-treated HHZ, an eliteindicarice cultivar widely grown in China[92].After removing the undesired mutations caused by EMS through backcrosses,HHZ carrying the Trp548Met mutation was registered as JTD-001 [84].JTD-001 showed no significant difference from HHZ in all the agricultural traits tested, indicating that the Trp548Met mutation had no detectable impact on rice growth and production [84].Compared with HHZ mutant carrying Ser627Asn mutation that confers resistance only to IMI herbicides,JTD-001 displayed resistance to all five families of ALS-inhibiting herbicides including IMI, SU,PTB, SCT, and TP, and the resistance index to IMI herbicides far exceeds the Ser627Asn mutant.These characteristics give Trp548Met several advantages over Ser627Asn in commercial applications.First,Trp548Met can be used to improve the safety of rotation crops.Ser627Asn mutation is resistant only to IMI herbicides which have a long turnover time.However, Trp548Met rice is resistant to many other types of ALS-inhibitors besides the IMI herbicides,and those with short turnover time and high herbicidal efficiency can be used in conjunction with the Trp548Met rice to solve the residue problem for rotation crops.Second, the high level of herbicide resistance of Trp548Met can protect the plants from high-dose herbicide toxicity which often happens in field application.Third,Trp548Met can increase the safety in hybrid rice production,because the heterozygous Trp548Met mutation can provide sufficient herbicide tolerance in field.At present, six Jietian rice varieties carrying Trp548Met have been released in China and quickly adopted by rice growers who use direct seeding for rice cultivation[93].Field experiments showed that the use of JTD-001 in combination with supporting herbicides effectively reduced the incidence of weedy rice to 0.07% in direct-seeded rice fields, in comparison with the incidence of weedy rice of 13.31%in the control field[93].The application of JTD-001 significantly reduced the cost in weed management and also increased the rice yield.As a result, the farmers who grew JTD-001 gained an extra income of$200-600 ha-1[93] which was very significant.

6.Future research and development of herbicide-resistant rice

6.1.Discover new genes and develop new herbicide-resistant rice

At present,glyphosate and glufosinate-resistant rice are mainly obtained through transgenic technology, and the safety of genetically modified rice is still controversial in the world, thus restricting the commercial application of transgenic herbicide-resistant rice.CRISPR/Cas9 technology and various gene editing systems developed based on this technology provide an effective way for modification of the rice endogenous genes and development of new herbicide-resistant rice[75-80].The genetically edited transgenic plants can be selfed or crossed to nontransgenic plants to separate the CRISPR/Cas9 transgene from the target mutation to obtain progenies without the transgene, and in nature, these progenies are the same as natural mutants and mutants generated by artificial mutagenesis.In many countries such as the Unite States, Brazil, Argentina and Canada, CRISPR/Cas9 edited crops were treated as nontransgenics.There are at least 262 weeds that have been reported to develop resistance to 23 types of herbicides[8].Comparison of the herbicide-target genes in the herbicideresistant and sensitive weeds will reveal if the mutations happen on the herbicide-target genes.Such information is helpful for designing gene-editing experiments to create mutations on the herbicide-target genes in rice in order to achieve improved resistance.By drawing on the mutation sites of herbicide-target genes in resistant weeds and other crops, we can test if mutations of the homologous genes in rice confer herbicide resistance using the CRISPR/Cas9 technology.If the level of resistance is not sufficiently high for field application, CRISPR/Cas9 editing of the mutant gene promoter can be further performed to elevate the mutant gene expression.Random editing of gene promoters has been shown to be a feasible approach to elevate gene expression[94].This would be one direction for future development of herbicide-resistant rice to different types of herbicides.In many cases, non-target-site resistance is responsible for the herbicide tolerance.The recently identifiedHIS1gene for triketone herbicides in rice is an example of such genes,which is likely to detoxify the herbicide in the plant [44].TheHIS1gene can be used for breeding of rice cultivars resistant to triketone herbicides.A number of metabolic genes such as cytochrome P450 monooxygenase,glycosyl transferase, and glutathione S-transferase are implicated in herbicide metabolic resistance in resistant weeds and crops[57].Some of these genes are highly induced in response to herbicide treatment,and for this characteristics,they were identified by differential gene expression experiments [57].Overexpression of these genes can elevate herbicide resistance in ectopic plants[57].These genes can be tested for herbicide resistance when overexpressed in rice.

The identification of new herbicide resistance genes and development of new herbicide-resistant rice are pressing, considering the rapid evolution of herbicide resistant weeds and weedy rice soon after the application of the nontransgenic herbicideresistant rice.Numerous cases of herbicide resistant weeds and weedy rice have been reported since the application of Clearfield Rice, which resulted from mutation of theALSgene in weeds or flow of the resistantALSgene from cultivated rice to weedy rice[2].It is expected that Jietian rice will soon face the same problem.The development of herbicide resistant weeds seriously challenges the continuing long term use of the ALS type of resistant rice technology.This problem asks for the development of new herbicide resistant rice that can tolerate different types of herbicides.By using different types of herbicide resistant rice alternately along with an appropriate stewardship, it can effectively slow down the development of resistant weeds.

6.2.Reducing the risks associated with herbicide application

Herbicide safety and residue effects on environment have always been public concerns.A herbicide must be safe and low toxic to human and animals before it can be approved for commercial use.Herbicides can directly or indirectly enter into soil after soil treatment or spay on plants, which may cause problems to the soil ecosystems.It has been shown that,after adding herbicides into the soil, the biomass of soil microorganisms was greatly reduced, the enzymatic activities in soil were inhibited, and the microbial community and microbial diversity were also changed[95].In addition, herbicide residues can also cause problems to rotation crops that are sensitive to the herbicide.Traditional methods such as tillage,irrigation,and adding biochar or humic acid can reduce herbicide residues in soil to some extent [96].At present,microbial degradation has become an effective method to handle the long-term herbicides.Degradation of IMI herbicide residues is mainly dependent on soil microbes, which was especially faster under aerobic conditions[97].The herbicide degradation is related to the soil types,physical and chemical properties,organic content,and microorganism varieties in the soil.Changing soil properties and applying degrading microorganisms to the soil can accelerate the degradation of herbicides to some extent.As to the safety of crop rotation, development of herbicide-resistant crops to the same type of herbicide is also an effective approach.For example,IMI-resistant corn, wheat, rapeseed, and sunflower have been developed that can be used as rotation crops with Clearfield rice and Jietian rice [12,93].Legume crops with a natural resistance to the IMI herbicides can also be used as rotation crops [98].As to the herbicide sensitive crops, safe intervals must be allowed for crop rotation.

Food safety associated with herbicide residues in the plant is also a public concern, which has caused hot debates on herbicide application especially after the International Agency for Research on Cancer (IARC) concluded in March 2015 that glyphosate is probably carcinogenic [99].To address the issue, development of herbicide resistant plants to safer herbicides is a way.Alternatively, development of herbicide resistant plants with increased activities to break down the herbicides can also provide a plausible solution.As to the herbicide resistant rice,experiments on residue detection and safety evaluation after high dose of IMI herbicide application showed that herbicide residues in brown rice,rice husk and straw were below the allowed dose at harvest.Thus, applying herbicides according to the GAD (Good Agricultural Practices)instructions is unlikely to induce dietary risk in rice.Nonetheless,molecular approaches to increase the plant abilities to quickly degrade the herbicides are still desirable because they cannot only enhance the plant resistance to herbicide but also reduce the herbicide residues for better food safety.In this hand,metabolic resistant genes that can break down herbicides to nontoxic compounds are particularly valuable.

6.3.Breeding of elite rice varieties suitable for different rice-growing regions

Currently, herbicide-resistant rice varieties in the world are mostly derived from Clearfield Rice CL161 carrying the Ser627Asn mutation,which is not an ideal resistant gene for its narrow resistance spectrum and the modest level of resistance to IMI.The application of Jietian Trp548Met mutation is at the infant stage due to the recent discovery of the gene.Only a few approved rice varieties are available at present carrying the Trp548Met mutation,and these rice varieties are suitable only to limited areas.The variable natural conditions in different rice-growing areas require the breeding of different varieties.Thus it is necessary to introduce the Trp548Met gene into different backgrounds to breeding new varieties suitable for different planting areas.Many rice genes for disease resistance, insect resistance, grain quality, aroma, stress tolerance, nutrient use efficiency, preferable plant architecture,etc., are available.It is necessary to select elite cultivars carrying these valuable agronomic genes as donor plants for further crossbreeding of new herbicide resistant varieties.Stacking of these valuable agronomic genes with Trp548Met gene can be assisted by marker-assisted selection, which will speed up the breeding of new varieties of high-yield, high-quality and resistance to diseases and herbicide.

CRediT authorship contribution statement

Man Jin:Writing - original draft.Lei Chen:Writing - original draft.Xing Wang Deng:Writing-review&editing,Funding acqusition, Methodology.Xiaoyan Tang:Writing - original draft, Writing - review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Natural Science Foundation of Guangdong Province (2018B030308008), National Natural Science Foundation of China(U1901203 and 31901532),Major Program of Guangdong Basic and Applied Research (2019B030302006), Shenzhen Commission on Innovation and Technology Programs JCYJ20180507181837997), and China Postdoctoral Science Foundation (2018 M633069 and 2019 M652920).