Genetic characterization of the AHAS mutant line K4 with resistance to AHAS-inhibitor herbicides in rapeseed (Brassica napus L.)

Abstract

It remains a great challenge to control weeds in rapeseed fields in China. Breeding herbicide-resistant rapeseed varieties and using corresponding herbicide formulations has become the most economical and effective way to control weeds in rapeseed field. Characterization of more herbicide-resistant genetic resources will provide opportunities for breeders to develop rapeseed herbicide-resistant varieties with good agronomic performance. Previously, we obtained the tribenuron methyl (TBM)-resistant mutant K4 from ZS9 (Brassica napus L.) through ethyl methyl sulfonate mutagenesis and TBM foliar-spray screening. In this study, the inheritance and molecular characterization of the mutant K4 are carried out. Genetic investigation indicated that the herbicide-resistance of the K4 was controlled by one dominant allele at a single nuclear gene locus. Molecular characterization showed that a single point substitution at position 535 from C to T in BnAHAS3 (BnAHAS3^535T), which resulted in a mutation at point 179 in BnAHAS3. The K4 showed a certain degree of resistance to TBM, bensulfuron methyl, and monosulfon sodium, which were 50, 30, and 5 times that of ZS9, respectively. AHAS enzyme assay, structural analysis of AHAS proteins, affinity detection between TBM and BnAHAS3 by surface plasmon resonance analysis, and the transgenic experiment in Arabidopsis using BnAHAS3^535T confirmed that BnAHAS3^535T endow the K4 with herbicides resistance. In addition, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS3^535T. In conclusion, our research identified and characterized one novel mutative AHAS allele in B. napus and enriched genetic resource for developing herbicide-resistant rapeseed cultivars.

Keywords

Rapeseed (Brassica napus L.) / Acetohydroxyacid synthase (AHAS) / Single-point mutation / Herbicides cross-resistance

References

[1] BlackshawRE, KanashiroD, MoloneyMM, CrosbyWL. Growth, yield and quality of canola expressing resistance to acetolactate synthase inhibiting herbicides. Can J Plant Sci, 1994, 74(4): 745-751.

[2] ChenY, HuangH, YuX, QiL. Chiral recognition of dextran sulfate with d- and l-cystine studied by multiwavelength surface plasmon resonance. Carbohyd Res, 2005, 340(12): 2024-2029.

[3] ChengHT, HaoMY, DingBL, MeiDS, WangWX, WangH, ZhouRJ, LiuJ, LiC, HuQ. Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system. Plant Biotechnol J, 2021, 19(1): 87-97.

[4] Doyle JJ, Doyle JL (1990) A rapid total DNA preparation procedure for fresh plant tissue. Focus 12:13–15. https://www.scienceopen.com/document?vid=6769c281-ab3f-4e1d-b01e-0414f9915561

[5] Duggleby RG, Pang SS (2000) Acetohydroxyacid synthase. J Biochem Mol Biol 33(1):1–36. http://espace.library.uq.edu.au/view/UQ:140764

[6] DugglebyRG, MccourtJA, GuddatLW. Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol Bioch, 2008, 46(3): 309-324.

[7] GaoJQ, PuHM, LongWH, HuML, QiCK, ZhangJF. Resistance of imidzolidone-resistant oilseed rape to nonselective herbicide imazethapyr. Jiangsu J Agr Sci, 2010, 26(6): 1186-1191.

[8] GarciaMD, NouwensA, LonhienneTG, GuddatLW. Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families. Proc Natl Acad Sci USA, 2017, 114(7): E1091-1100.

[9] GhioC, RamosML, AltieriE, BulosM, SalaCA. Molecular characterization of Als1, an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean. Theo Appl Genet, 2013, 126(12): 2957-2968.

[10] GuoY, ChengL, LongWH, GaoJQ, ZhangJF, ChenS, PuHM, HuML. Synergistic mutations of two rapeseed AHAS genes confer high resistance to sulfonylurea herbicides for weed control. Theor Appl Genet, 2020, 133(10): 2811-2824.

[11] GuoY, LiuCL, LongWH, GaoJQ, ZhangJF, ChenS, PuHM, HuML. Development and molecular analysis of a novel acetohydroxyacid synthase rapeseed mutant with high resistance to sulfonylurea herbicides. Crop J, 2022, 10(1): 56-66.

[12] HallTA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser, 1999, 41(41): 95-98.

[13] HattoriJ, BrownD, MouradG, LabbéH, OuelletT, SunoharaG, RutledgeR, KingJ, MikiB. An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance. Mol Gen Genet, 1995, 246(4): 419-425.

[14] HaughnGW, SomervilleCR. A mutation causing imidazolinone resistance maps to the csr1 locus of Arabidopsis thaliana. Plant Physiol, 1990, 92(4): 1081-1085.

[15] HeapI. Global perspective of herbicide-resistant weeds. Pest Manag Sci, 2014, 7091306.

[16] Hu ML, Pu HM, Gao JQ, Long WH, Qi CK, Zang JF, Chen S (2012) Inheritance and gene cloning of an ALS inhabiting herbicide-resistant mutant line M9 in Brassica napus. Sci Agric Sin 45(20):4326–4334. https://doi.org/10.3864/j.issn.0578-1752.2012.20.022

[17] HuML, PuHM, KongLN, GaoJQ, LongWH, ZhangJF, QiCK. Molecular characterization and detection of a spontaneous mutation conferring imidazolinone resistance in rapeseed and its application in hybrid rapeseed production. Mol Breeding, 2015, 3546.

[18] HuML, PuHM, GaoJQ, LongWH, ChenF, ZhouXY, ZhangW, PengQ, ChenS, ZhangJF. Inheritance and molecular characterization of resistance to AHAS-inhibiting herbicides in rapeseed. J Integr Agr, 2017, 16(11): 2421-2433.

[19] HuML, PuHM, ZhangJF. Research advances of rapeseed germplasms with ALS-inhibiting herbicides resistance in China. Chin J Oil Crop Sci, 2018, 40(5): 679-686.

[20] HuML, ChengL, GuoY, LongWH, GaoJQ, PuHM, ZhangJF, ChenS. Development and application of the marker for imidazolinone-resistant gene in Brassica napus. Acta Agron Sin, 2020, 46(10): 1639-1646.

[21] Huang QX, Lv JY, Sun YY, Wang HM, Guo Y, Qu GP, Hu SW (2020) Inheritance and molecular characterization of a novel mutated AHAS gene responsible for the resistance of AHAS-inhibiting herbicides in rapeseed (Brassica napus L.). Int J Mol Sci 21(4):1345. https://doi.org/10.3390/ijms21041345

[22] IBM Corp (2013) IBM SPSS statistics for windows, version 20.0. Armonk: IBM Corp.

[23] KaracaAC, LowN, NickersonM. Emulsifying properties of canola and flaxseed protein isolates produced by isoelectric precipitation and salt extraction. Food Res Int, 2011, 44(9): 2991-2998.

[24] KleschickWA, CostalesMJ, DunbarJE, MeikleRW, MonteWT, PearsonNR, SniderSW, VinogradoffAP. New herbicidal derivatives of 1,2,4-triazolo [1,5-a] pyrimidine. Pestic Sci, 1990, 29(4): 341-355.

[25] LiH, LiJ, ZhaoB, WangJ, YiL, LiuC, WuJ, KingGJ, LiuK. Generation and characterization of tribenuron-methyl herbicide-resistant rapeseed (Brasscia napus) for hybrid seed production using chemically induced male sterility. Theor App Genet, 2015, 128: 107-118.

[26] LonhienneT, ChengY, GarciaMD, HuSH, YuS, LowYS, SchenkG, WilliamsCM, GuddatLW. Structural basis of resistance to herbicides that target acetohydroxyacid synthase. Nat Commun, 2022, 133368.

[27] LvJY, HuangQX, SunYY, QuGP, GuoY, ZhangXJ, ZhaoHX, HuSW. Male sterility of an AHAS-mutant induced by tribenuron-methyl solution correlated with the decrease of AHAS activity in Brassica napus L. Front Plant Sci, 2018, 91014.

[28] MccourtJA, DugglebyRG. Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids. Amino Acids, 2006, 31(2): 173-210.

[29] MendelsohnAR, BrentR. Protein interaction methods-toward an endgame. Science, 1999, 284(5422): 1948-1950.

[30] QuGP, SunYY, PangHX, QiangWU, WangFL, HuSW. EMS mutagenesis and ALS-inhibitor herbicide-resistant mutants of Brassica napus L. Chin J Oil Crop Sci, 2014, 36(1): 25-31.

[31] RavindranN, KumarS, YashiniM, RajeshwariS, MamathiCA, ThirunavookarasuNS, SunilCK. Recent advances in Surface Plasmon Resonance (SPR) biosensors for food analysis: a review. Crit Re Food Sci Nut, 2021, 63(8): 1055-1077.

[32] RayTB. Site of action of chlorsulfuron: inhibition of valine and isoleucine biosynthesis in plants. Plant Physiol, 1984, 75(3): 827-831.

[33] RutledgeRG, QuelletT, HattoriJ, MikiBL. Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family. Mol Gen Genet, 1991, 229(1): 31-40.

[34] Santel HJ, Bowden BA, Sorensen VM, Mueller KH (1999) Flucarbazone-sodium - a new herbicide for the selective control of wild oat and green foxtail in wheat. Proc West Soc Weed Sci 76:545–546. https://www.researchgate.net/publication/284648743

[35] SchaggerH, JagowGV. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Ana Biochem, 1987, 166(2): 368-379.

[36] SchusterSC, SwansonRV, AlexLA, BourretRB, SimonMI. Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance. Nature, 1993, 365(6444): 343-347.

[37] SeefeldtSS, JensenJE, FuerstEP. Log-logistic analysis of herbicide dose-response relationships. Weed Technol, 1995, 9(2): 218-227.

[38] ShanerDL, AndersonPC, StidhamMA. Imidazolinones: potent inhibitors of acetohydroxyacid synthase. Plant Physiol, 1984, 76(2): 545-546.

[39] ShenGY, GaoY, DaiDS, CuiJ, LiuYM, PeiLP. Kinetics study of chiral recognition between protein and amino acid enantiomers by surface plasmon resonance. Chem J Chinese U, 2011, 32(8): 1709-1713.

[40] ShiJ, YuH, FuY, WangT, ZhangY, HuangJ, LiS, ZhengT, NiX, ZhaoJ. Development and validation of functional kompetitive allele-specific PCR markers for herbicide resistance in Brassica napus. Front Plant Sci, 2023, 141213476.

[41] SubramanianMV, HungHY, DiasJM, MinerVW, ButlerJH, JachettaJJ. Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide. Plant Physiol, 1990, 94(1): 239-244.

[42] SunYY, QuGP, HuangQX, LvJY, GuoYWuHS. SNP markers for acetolactate synthase genes from tribenuron-methyl resistant mutants in Brassica napus L. Chin J Oil Crop Sci, 2015, 37(5): 589-595.

[43] SwansonEB, HerrgesellMJ, ArnoldoM, SippellDW, WongRSC. Microspore mutagenesis and selection: Canola plants with field tolerance to the imidazolinones. Theor App Genet, 1989, 78(4): 525-530.

[44] TanS, EvansRR, DahmerML, SinghBK, ShanerDL. Imidazolinone-tolerant crops: history, current status and future. Pest Manag Sci, 2005, 61(3): 246-257.

[45] TonnemakerKA, AuldDL, ThillDC, Mallory-SmithCA, EricksonDA. Development of sulfonylurea-resistant rapeseed using chemical mutagenesis. Crop Sci, 1992, 32(6): 1387-1391.

[46] Tranel PJ, Wright TR, Heap IM (2024) Mutations in herbicide-resistant weeds to Inhibition of Acetolactate Synthase. Online http://www.weedscience.com

[47] WuJ, ChenC, XianG, LiuD, WangY. Engineering herbicide-resistant oilseed rape by CRISPR/Cas9-mediated cytosine base-editing. Plant Biotechnol J, 2020, 18(9): 1857-1859.

[48] XinXY, QuGP, ZhangR, PangHX, WuQ, WangFL, HuSW. Identification of the tribenuron-methyl tolerance in different rapeseed genotypes. Acta Agriculturae Boreali-Occidentalis Sinica, 2014, 23(7): 68-74.

[49] YuQ, PowlesSB. Resistance to AHAS inhibitor herbicides: current understanding. Pest Manag Sci, 2014, 70(9): 1340-1350.

[50] ZhangX, HenriquesR, LinSS, NiuQW, ChuaNH. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc, 2006, 1(2): 641-646.

[51] Zhang HJ, Liu X, Zhang J, Cui HL, Zhang CX, Zhu WD (2008) Registration and use of herbicides in rapeseed fields in China. Sci Technol Innov Her 15:252–253. https://doi.org/10.16660/j.cnki.1674-098x.2008.15.174

RIGHTS & PERMISSIONS

The Author(s)