Inheritance and Molecular Characterization of a Novel Mutated AHAS Gene Responsible for the Resistance of AHAS-Inhibiting Herbicides in Rapeseed (Brassica napus L.)

The use of herbicides is an effective and economic way to control weeds, but their availability for rapeseed is limited due to the shortage of herbicide-resistant cultivars in China. The single-point mutation in the acetohydroxyacid synthase (AHAS) gene can lead to AHAS-inhibiting herbicide resistance. In this study, the inheritance and molecular characterization of the tribenuron-methyl (TBM)-resistant rapeseed (Brassica napus L.) mutant, K5, are performed. Results indicated that TBM-resistance of K5 was controlled by one dominant allele at a single nuclear gene locus. The novel substitution of cytosine with thymine at position 544 in BnAHAS1 was identified in K5, leading to the alteration of proline with serine at position 182 in BnAHAS1. The TBM-resistance of K5 was approximately 100 times that of its wild-type ZS9, and K5 also showed cross-resistance to bensufuron-methyl and monosulfuron-ester sodium. The BnAHAS1544T transgenic Arabidopsis exhibited higher TBM-resistance than that of its wild-type, which confirmed that BnAHAS1544T was responsible for the herbicide resistance of K5. Simultaneously, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS1544T. In addition, a method for the fast screening of TBM-resistant plants at the cotyledon stage was developed. Our research identified and molecularly characterized one novel mutative AHAS allele in B. napus and laid a foundation for developing herbicide-resistant rapeseed cultivars

Image_3_Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L..JPEG

<p>Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1^544T), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L^-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be^(-^KX⁾) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1^544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.</p

Image_2_Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L..JPEG

<p>Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1^544T), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L^-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be^(-^KX⁾) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1^544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.</p

Image_4_Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L..JPEG

<p>Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1^544T), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L^-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be^(-^KX⁾) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1^544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.</p

Image_5_Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L..JPEG

<p>Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1^544T), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L^-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be^(-^KX⁾) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1^544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.</p

Light-induced LLPS of the CRY2/SPA1/FIO1 complex regulating mRNA methylation and chlorophyll homeostasis in <i>Arabidopsis</i>

Light regulates chlorophyll homeostasis and photosynthesis via various molecular mechanisms in plants. The light regulation of transcription and protein stability of nuclear-encoded chloroplast proteins have been extensively studied, but how light regulation of mRNA metabolism affects abundance of nuclear-encoded chloroplast proteins and chlorophyll homeostasis remains poorly understood. Here we show that the blue light receptor cryptochrome 2 (CRY2) and the METTL16-type m6A writer FIONA1 (FIO1) regulate chlorophyll homeostasis in response to blue light. In contrast to the CRY2-mediated photo-condensation of the mRNA adenosine methylase (MTA), photoexcited CRY2 co-condenses FIO1 only in the presence of the CRY2-signalling protein SUPPRESSOR of PHYTOCHROME A (SPA1). CRY2 and SPA1 synergistically or additively activate the RNA methyltransferase activity of FIO1 in vitro, whereas CRY2 and FIO1, but not MTA, are required for the light-induced methylation and translation of the mRNAs encoding multiple chlorophyll homeostasis regulators in vivo. Our study demonstrates that the light-induced liquid–liquid phase separation of the photoreceptor/writer complexes is commonly involved in the regulation of photoresponsive changes of mRNA methylation, whereas the different photo-condensation mechanisms of the CRY/FIO1 and CRY/MTA complexes explain, at least partially, the writer-specific functions in plant photomorphogenesis