A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

Background Herbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds.Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches.At present,no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism(GMO)regulation law.We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate(EMS)mutagenesis,offering an alternative choice for weed management.Results Seeds of an elite cotton cultivar Lumianyan 37(Lu37)were treated with EMS,and a mutant Lu37-1 showed strong tolerance to imidazolinone(IMI)herbicides was identified.A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase(ALS)(Gh_D10G1253)in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton.The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species.Cleaved amplified polymorphic sequence(CAPS)markers were developed to identify the ALS mutant allele.The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.Conclusion The nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identi-fied mutation in cotton.This substitution mutation has also been identified in the orthologous ALS genes in other crops.This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.

Activity of Acetolactate Synthase from Maize (Zea mays L.) as Influenced by Chlorsulfuron and Tribenuron-methyl

Study on relative sensitivity of maize (Zea mays L.) Nongda108 and Nongda3138 to sulfony-lurea herbicide chlorsulfuron and tribenuron-methyl using maize taproot length by sand bioassy indicated that, Nongda3138 had higher tolerance to chlorsulfuron and tribenuron-methyl than Nongda108 did. Chlorsulfuron had stronger growth inhibition to maize Nongda108 and Nongda3138 than tribenuron-methyl did. Study on target enzyme of sulfonylurea herbicide acetolactate synthase (ALS) showed that, chlorsulfuron and tribenuron-methyl inhibited ALS in vitro strongly, and non-competitively. In the same concentration of inhibitors, chlorsulfuron had stronger ALS activity inhibition than tribenuron-methyl did. Lower level of chlorsulfuron and tribenuron-methyl has no ALS activity inhibition in vivo, the ALS inhibition only occurred in the condition of high concentration of chlorsulfuron and tribenuron-methyl in vivo.

Trp_(548)Met mutation of acetolactate synthase in rice confers resistance to a broad spectrum of ALS-inhibiting herbicides

Herbicide resistance in crop plants is valuable for integrated weed management in agriculture. Herbicide resistant rice, in particular, is important to management of weedy rice, a close relative of cultivated rice and a noxious weed prevalent in rice fields that remains challenging to farmers worldwide. Herbicide resistant plants can be obtained through transgenic approach or by mutagenesis of regular plant and screening of mutants with elevated resistance to herbicide. In this study, we conducted ethyl methyl sulfonate mutagenesis(EMS) to elite indica cultivar Huanghuazhan(HHZ) and screened for mutants resistant to imazapic, a herbicide that can inhibit the acetolactate synthase(ALS) in plants. We obtained three mutants of Os ALS gene that have not been reported previously in rice. One of the mutants, with Trp_(548) changed to Met(W_(548)M), was analyzed in more details in this study. This mutation had no negative effect on the plant physiology and morphology as well as rice yield. Compared with the imidazolinone-resistant mutant S_(627)N(Ser_(627) changed to Asn) that has been deployed for Clearfield rice development, W_(548)M mutant showed high levels of resistance to a broad spectrum of five families of ALSinhibiting herbicides, in addition to a higher level of resistance to herbicides of the imidazolinone family.The herbicide-resistance was stably inherited by crossing into other rice lines. Thus, the W_(548)M mutation provides a valuable resource for breeding of herbicide resistant rice and weed management.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

Tribenuron-Methyl Induces Male Sterility through Anther-Specific Inhibition of Acetolactate Synthase Leading to Autophagic Cell Death

Tribenuron-methyl （TM） is a powerful sulfonylurea herbicide that inhibits branched-chain amino acid （BCAA） biosynthesis by targeting the catalytic subunit （CSR1） of acetolactate synthase （ALS）. Selective in- duction of male sterility by foliar spraying of TM at low doses has been widely used for hybrid seed produc- tion in rapeseed （Brassica napus）; however, the underlying mechanism remains unknown. Here, we report greater TM accumulation and subsequent stronger ALS inhibition and BCAA starvation in anthers than in leaves and stems after TM application. Constitutive or anther-specific expression of csrl-lD （a CSR1 mutant） eliminated anther-selective ALS inhibition and reversed the TM-induced male sterile phenotype in both rapeseed and Arabidopsis. The results of TM daub-stem experiments, combined with the observations of little TM accumulation in anthers and reversion of TM-induced male sterility by targeted expression of the TM metabolism gene Bel in either the mesophyll or phloem, suggested that foliar-sprayed TM was polar-transported to anthers mainly through the mesophyll and phloem. Microscopy and immunoblotting revealed that autophagy, a bulk degradation process induced during cell death, was elevated in TM-induced male sterile anthers and by anther-specific knockdown of ALS. Moreover, TM-induced pollen abortion was significantly inhibited by the autophagy inhibitor 3-MA. These data suggested that TM was polar-transported to anthers, resulting in BCAA starvation via anther-specific ALS inhibition and, ulti- mately, autophagic cell death in anthers.

Study on Mutations in ALS for Resistance to Tribenuron-Methyl in Galium aparine L.

In recent years, Galium aparine L. has not been controlled by tribenuron-methyl in major Chinese winter wheat fields. The objective of this study is to understand the molecular basis of the resistance mechanism to tribenuron-methyl in G. aparine and to find the specific mutation sites in amino acid sequence of acetolactate synthase （ALS） in the resistant biotype of G. aparine. Fragments that encode the ALS were amplified and cloned from susceptible （S） and resistant （R） biotypes of G. aparine to tribenuron-methyl and sequenced subsequently. The result showed that the nucleotide sequence of Rbiotype of G. aparine differed from that of the S biotype with three amino acid substitutions, of which, the amino acid substitution of Trp57-4 （TGG） to Gly （GGG） is located in the highly conserved region Domain B. The substitution of Trp574 might be responsible for the resistance to tribenuron-methyl in the R-biotype of G. aparine.

Herbicide activity of monosulfuron and its mode of action

Monosulfuron was developed for weed control in the field of wheat(Triticum, aestivum L.) and millet(Panicum miliaceum L.) with the application rate ranging from 15 to 60 g ai/hm2. Herbicidal activity of monosulfuron was evaluated systematically by bioassay using maize(Zea mays L.) taproot as indicator and weed fresh weight of Acalypha australis L. and Echinochloa phyllopogon. Maize CAU 3138 was the most tolerant cultivars to monosulfuron with IC_ 50(concentration of 50% inhibition) of 85 μg/kg, Yedan 13 was one of the most sensitive cultivars to monosulfuron with IC_ 50 of 6.4 μg/kg. Monosulfuron inhibited the growth of Acalypha australis L. strongly comparing with that of Echinochloa phyllopogon. Monosulfuron was a good acetolactate synthase(ALS) inhibitor in vitro, the I_ 50(50% of inhibition) of monosulfuron, chlorsulfuron, tribenuron-methyl and nicosulfuron for CAU 3138 were 32, 2, 19 and 26 nmol/L respectively, for Yedan 13 the I_ 50 were 15, 3, 17 and 65 nmol/L respectively. In vivo ALS inhibition occurred only in higher concentration of 4 sulfonylurea herbicide tested. Comparison study of this test indicated that the mode of action of monosulfuron was the same as that of other sulfonylurea herbicides such as chlorsulfuron, tribenuron-methyl and nicosulfuron, they were all inhibitors targeted at the ALS.

Effect of Chlorimuron-Ethyl on Biochemical Mechanism in Tolerant Sugar Beet

Effect of chlorimuron-ethyl on biochemical mechanism in tolerant sugar beet was investigated to provide basic data on using the tolerant genotype properly. Tolerant sugar beet was used to analyze its biochemical mechanism under chlorimuron- ethyl stress with frame culture in field and water culture. Glutathione S-transferase （GST） activity of leaves in tolerant sugar beet was remarkably increased as chlorimuron-ethyl was preemergence applicated at 0.5 and 1.5 g a.i. hw1, at the same time glutathione （GSH） content increased 50.0-490.1 p.g g-~. GST activity of sensitive sugar beet decreased 122.6 U mg-~ min-1 compared with tolerant sugar beet and GSH content only increased to 7.4 p.g g-~ at chlorimuron-ethyl 0.5 g a.i. ha-~ in sensitive sugar beet. The higher GST activity and GSH content conjugated chlorimuron-ethyl absorbed in tolerant sugar beet and made it lost activity. Acetolactate synthases （ALS） activity of the tolerant sugar beet increased to 62.5 and 70.6%, respectively in seedling and leaf growth period, at the same time ALS activity of the sensitive variety was decreased to 36.8 and 64.8%, respectively. The rapidly increased GST activity, GSH content, and ALS, the target enzyme activity were the important pathways for enduring chlorimuron-ethyl in tolerant sugar beet.

Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing

Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.