Acetohydroxyacid Synthase Gene Research Articles

Allelic interactions defining Raphanus raphanistrum AHAS resistance level: strong vs weak target-site AHAS resistance alleles.

Chlorsulfuron resistance and genetic dominance was evaluated in Raphanus raphanistrum genotypes homozygous (122-RR, 376-RR), heterozygous (122-RS, 376-RS) and compound heterozygous (122-R/376-R) for the target-site resistance mutations Ala-122-Tyr and Asp-376-Glu in the AHAS (acetohydroxyacid synthase) gene. At the AHAS level, 122-RR and 122-RS plants exhibited significantly higher I50 values than 376-RR and 376-RS plants, respectively. However, plants of the compound heterozygous genotype (122-R/376-R), showed no difference in AHAS activity compared to the 122-RS genotype but lower activity than the 122-RR genotype, and showed a nearly 400-fold greater I50 value than both genotypes (376-RR and 376-RS) carrying the 376-Glu allele. At the whole-plant level, 100% survival was observed for 122-RR plants at the highest chlorsulfuron dose of 640 g ha-1, yet 376-RR plants showed no survival at 380 g ha-1. Thus, this survival difference resulted in different median lethal dose (LD50) estimates [>640 (122-RR) versus 330 g ha-1(376-RR)]. The effect of chlorsulfuron in reducing aboveground growth of surviving plants also was markedly lower for the homozygous 122-RR (GR50 = 566 g ha-1) than for 376-RR plants (GR50 = 66). Heterozygous plants carrying the 122-Tyr allele (122-RS) exhibited two- and five-fold higher LD50 values than both homozygous and heterozygous plants carrying the 376-Glu allele (376-RR, 376-RS), respectively. Along the difference in plant survival, 122-RS plants also showed four-fold higher GR50 than both 376-RR and 376-RS plants. Survival of plants with the compound heterozygous genotype (122-R/376-R) under increasing chlorsulfuron doses was similar to 122-RR or 122-RS genotypes. However, this compound heterozygous genotype showed two- and six-fold higher LD50 values than 376-RR or 376-RS genotypes, respectively. However, both resistance 122-Tyr and 376-Glu alleles were dominant or nearly dominant over the wild-type susceptible alleles (ALA-122 and ASP-376), and the resistance 122-Tyr allele was dominant over the 376-Glu allele. These results broaden our understanding of AHAS target-site resistance in R. raphanistrum and strengthens the hypothesis that the AHAS 122-Tyr allele corresponds to a stronger target-site resistance allele than the 376-Glu allele. © 2024 Society of Chemical Industry.

Intragenic Agrobacterium-mediated gene transfer mimics micro-translocations without foreign DNA

Main conclusionAgrobacterium-mediated transformation of Nicotiana tabacum, using an intragenic T-DNA region derived entirely from the N. tabacum genome, results in the equivalence of micro-translocations within genomes.Intragenic Agrobacterium-mediated gene transfer was achieved in Nicotiana tabacum using a T-DNA composed entirely of N. tabacum DNA, including T-DNA borders and the acetohydroxyacid synthase gene conferring resistance to sulfonylurea herbicides. Genomic analysis of a resulting plant, with single locus inheritance of herbicide resistance, identified a single insertion of the intragenic T-DNA on chromosome 5. The insertion event was composed of three N. tabacum DNA fragments from other chromosomes, as assembled on the T-DNA vector. This validates that intragenic transformation of plants can mimic micro-translocations within genomes, with the absence of foreign DNA.

Target and nontarget mechanisms of AHAS inhibitor cross-resistance patterns in Cyperus difformis

Weed resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides has been a critical issue for rice growers worldwide since the early 1990's. In California, resistance to bensulfuron-methyl was first detected in Cyperus difformis in 1993. Since then, populations of most major weeds of rice in California have been reported to show resistance to at least one AHAS inhibitor. We sought to describe the magnitude and mechanisms of AHAS inhibitor cross-resistance in California populations of C. difformis. Sixty-two populations were collected and screened for cross-resistance to bensulfuron-methyl (BEN), halosulfuron-methyl (HAL), bispyribac‑sodium (BIS), and penoxsulam (PEN), revealing six major patterns of cross-resistance. Representative C. difformis populations from each cross-resistance pattern were then subjected to dose-response, cytochrome P450 inhibition, AHAS gene sequencing, and metabolic studies with the same herbicides as in the screening. Dose-response confirmed the detected resistances in the representative populations, and suggested that the majority of observed resistance was dose-dependent. Cytochrome P450 inhibition via malathion revealed evidence of increased metabolic activity in resistant populations to BEN, BIS, and PEN. AHAS gene sequencing revealed amino acid substitutions in five of six populations: R3 (Pro197-Ser), R4 (Pro97-His), R10 (Asp376), R41 (Ala122-Asn), and R18 (Trp574-Leu). Metabolic studies confirmed evidence of increased activity of cytochrome P450s in all populations. Metabolic BEN and HAL analysis did not yield similar results to malathion inhibition, suggesting different P450's or other pathways. Taken together, the results of the studies confirm the complexity of AHAS inhibitor cross-resistance in C. difformis, and the presence of both target-site and metabolic resistance in most of the representative populations underscores the importance of proper herbicide selection, rotation, and scouting in fields.

Genetic origin and expression patterns of acetohydroxyacid synthase multigene family in Brassica juncea and B. carinata and their progenitors.

Acetohydroxyacid synthase (AHAS), the key enzyme in the branched-chain amino acids leucine, isoleucine, and valine biosynthesis pathway, has gained intensive investigation because it is the target of five different AHAS herbicides widely used to control weeds in farmland. In the present study, the AHAS gene family in Brassica juncea and B. carinata and their progenitor species was characterized in combination with bioinformatics, gene-specific PCR and qRT-PCR analyses. The results indicated that B. juncea contains four AHAS genes, of them, BjuAHAS3 and BjuAHAS4 originated from the A genome donor of B. rapa, whereas BjuAHAS6 and BjuAHAS7 from the B genome donor of B. nigra. BjuAHAS3 and BjuAHAS6 are predicted to be functional and constitutively expressed in all vegetative and reproductive tissues in the tested B. juncea accessions. B. carinata contains five AHAS genes, of them, BcaAHAS1, BcaAHAS2, and BcaAHAS5 originated from the C genome donor of B. oleracea, whereas BcaAHAS6 and BcaAHAS7 came from the B genome donor of B. nigra. BcaAHAS1, BcaAHAS2, and BcaAHAS6 are predicted to be functional. BcaAHAS1 and BcaAHAS6 are constitutively expressed in all vegetative and reproductive tissues in the tested B. carinata accessions, however, BcaAHAS2 is mainly expressed in siliques. In addition, translocation events for the AHAS1, AHAS2, and AHAS7 genes occurred when the three amphidiploids species B. napus, B. juncea, and B. carinata were formed by hybridization of their respective diploid species. The findings in this study will provide important basic information for the breeding of herbicide-resistant varieties in B. juncea and B. carinata.

Development and molecular analysis of a novel acetohydroxyacid synthase rapeseed mutant with high resistance to sulfonylurea herbicides

With the increasing promotion of simplified rapeseed cultivation in recent years, the development of cultivars with high resistance to herbicides is urgently needed. We previously developed M342, which shows sulfonylurea herbicide resistance, by targeting acetohydroxyacid synthase (AHAS), a key enzyme in branched-chain amino acid synthesis. In the present study, we used a progeny line derived from M342 for an additional round of ethyl methane sulfonate mutagenesis, yielding the novel mutant DS3, which harbored two mutations in AHAS genes and showed high sulfonylurea resistance. One mutation was the substitution Trp574Leu, as in M342, according to Arabidopsis protein sequencing. The other site was a newly recognized substitution, Pro197Leu. A KASP marker targeting Pro197Leu was developed and reliably predicted the response to sulfonylurea herbicides in the F2 population. The combination of Trp574Leu and Pro197Leu in DS3 produced a synergistic effect that greatly increased herbicide resistance. Analysis of the protein structures of AHAS1 and AHAS3 in wild-type and single-gene mutant plants revealed three-dimensional protein conformational changes that could account for differences in herbicide resistance characteristics including toxicity tolerance, AHAS enzyme activity, and AHAS gene expression.

A Cisgenic Approach in the Transformation of Bread Wheat cv. Saratovskaya 29 with Class I Chitinase Gene

Background: Bread wheat is one of the major crops grown worldwide, showing high demand for new varieties with traits such as pathogen resistance. As the public acceptance of transgenic plants remains low, a novel approach – cisgenesis – is being developed to introduce the genes from the same or closely related species. Objective: This study presents a cisgenic approach used for the transformation of wheat with class I chitinase gene derived from T. aestivum cv. Stepnaya 15, co-transformed with acetohydroxyacid synthase gene that provides tolerance to imidazolinone herbicides. Methods: Calli from immature embryos of spring bread wheat Triticum aestivum cv. Saratovskaya 29 were used for co-transformation with two independent Minimal Expression Units (MEUs): class I chitinase and Acetohydroxyacid Synthase (AHAS) gene. For identification of cisgenic plants, genomic DNA was extracted from the leaves of imazethapyr-resistant regenerant plants at the plantlets stage and screened by polymerase chain reaction. The efficiency of transformation was calculated as the relation of regenerated plants with chitinase gene insert to the total number of calli in the experiment. Results: The average transformation efficiency in four series of experiments (total number of calli - 2299) was found to be 1.84% (ranging from 0.3% to 3.4%), while total co-transformation efficiency reached 87.93%. Conclusion: The high efficiency of co-transformation in the experiment promotes it as a very useful technique for the production of wheat lines, free of the selectable marker gene. To our knowledge, this is the first report of cisgenic bread wheat, where both target and selectable genes are derived from wheat.

Molecular basis of cross-resistance to acetohydroxy acid synthase-inhibiting herbicides in Sagittaria trifolia L.

Acetohydroxy acid synthase (AHAS)-inhibiting herbicides are one of the most commonly used herbicides for controlling the growth of Sagittaria trifolia L. in paddy fields in Northeastern China. In this study, we collected five suspected resistant populations of S. trifolia (R1-R5) from three different provinces of Northeastern China. The results of whole-plant bioassays revealed that those populations showed high level of resistance to bensulfuron-methyl with resistance index (GR50 R/S) ranging from 39.90 to 88.50. The results of AHAS-activity assays were consistent with the results of the whole-plant bioassays. The AHAS gene analysis showed that R2 and R3 populations contained Pro-197-Leu mutations that were highly resistant to penoxsulam; R1 and R4 populations contained Pro-197-Ser mutations that were highly resistant to bispyribac‑sodium; R5 population contained Trp-574-Leu mutation that showed high resistance to IMI, PT, PTB and SU herbicides. The AHAS with resistance mutations showed less sensitivity to feedback inhibition by BCAAs and R genotypes had increased free BCAAs

Marker Assisted Development and Characterization of Herbicide Tolerant Near Isogenic Lines of a Mega Basmati Rice Variety, \u201cPusa Basmati 1121\u201d

BackgroundDirect-seeded rice (DSR) is a potential technology for sustainable rice farming as it saves water and labor. However, higher incidence of weed under DSR limits productivity. Therefore, there is a need to develop herbicide tolerant (HT) rice varieties.ResultsWe used marker assisted backcross breeding (MABB) to transfer a mutant allele of Acetohydroxy acid synthase (AHAS) gene, which confers tolerance to imidazolinone group of herbicides from the donor parent (DP) “Robin” into the genetic background of an elite popular Basmati rice variety, Pusa Basmati 1121 (PB 1121). Foreground selection was done using the AHAS gene linked Simple Sequence Repeat (SSR) marker RM6844 and background selection was performed using 112 genome-wide SSR markers polymorphic between PB 1121 and Robin. Phenotypic selection for agronomic, Basmati grain and cooking quality traits in each generation was carried out to improve the recovery of recurrent parent phenome (RPP). Finally, a set of 12 BC4F4 near isogenic lines (NILs), with recurrent parent genome (RPG) recovery ranging from 98.66 to 99.55% were developed and evaluated. PB 1121-HT NILs namely 1979-14-7-33-99-10, 1979-14-7-33-99-15 and 1979-14-7-33-99-66 were found superior to PB 1121 in yield with comparable grain and cooking quality traits and herbicide tolerance similar to Robin.ConclusionOverall, the present study reports successful development of HT NILs in the genetic background of popular Basmati rice variety, PB 1121 by introgression of mutated AHAS allele. This is the first report on the development of HT Basmati rice. Superior NILs are being evaluated in the national Basmati trials, the release of which is likely to provide a viable option for the adoption of DSR technology in Basmati rice cultivation.

Ser-653-Asn substitution in the acetohydroxyacid synthase gene confers resistance in weedy rice to imidazolinone herbicides in Malaysia.

The continuous and sole dependence on imidazolinone (IMI) herbicides for weedy rice control has led to the evolution of herbicide resistance in weedy rice populations across various countries growing IMI herbicide-resistant rice (IMI-rice), including Malaysia. A comprehensive study was conducted to elucidate occurrence, level, and mechanisms endowing resistance to IMI herbicides in putative resistant (R) weedy rice populations collected from three local Malaysian IMI-rice fields. Seed bioassay and whole-plant dose-response experiments were conducted using commercial IMI herbicides. Based on the resistance index (RI) quantification in both experiments, the cross-resistance pattern of R and susceptible (S) weedy rice populations and control rice varieties (IMI-rice variety MR220CL2 and non-IMI-rice variety MR219) to imazapic and imazapyr was determined. A molecular investigation was carried out by comparing the acetohydroxyacid synthase (AHAS) gene sequences of the R and S populations and the MR220CL2 and MR219 varieties. The AHAS gene sequences of R weedy rice were identical to those of MR220CL2, exhibiting a Ser-653-Asn substitution, which was absent in MR219 and S plants. In vitro assays were conducted using analytical grade IMI herbicides of imazapic (99.3%) and imazapyr (99.6%) at seven different concentrations. The results demonstrated that the AHAS enzyme extracted from the R populations and MR220CL2 was less sensitive to IMI herbicides than that from S and MR219, further supporting that IMI herbicide resistance was conferred by target-site mutation. In conclusion, IMI resistance in the selected populations of Malaysian weedy rice could be attributed to a Ser-653-Asn mutation that reduced the sensitivity of the target site to IMI herbicides. To our knowledge, this study is the first to show the resistance mechanism in weedy rice from Malaysian rice fields.

Synergistic mutations of two rapeseed AHAS genes confer high resistance to sulfonylurea herbicides for weed control.

A double mutant 5N of rapeseed was obtained with a synergistic effect of high resistance to sulfonylurea herbicide. Excellent weed control was observed in Ning R201 created by 5N resources. Sulfonylurea herbicides, which inhibit acetohydroxyacid synthase (AHAS), have become the most widely used herbicides worldwide. However, weed control in rapeseed crop production remains challenging in China due to the shortage of available herbicide-resistant cultivars. In this study, we developed a rapeseed line (PN19) with sulfonylurea herbicide resistance through seed mutagenesis. Molecular analysis revealed a Trp-574-Leu mutation in BnAHAS1-2R of PN19 according to the sequence of Arabidopsis thaliana, and an allele-specific cleaved amplified polymorphic sequence marker was developed to target the point mutation. A double mutant (5N) with very high sulfonylurea resistance was then created through pyramiding two mutant genes of PN19 and M342 by molecular marker-assisted selection. Herbicide resistance identification, toxicology testing, and an in vitro enzyme activity assay of AHAS in 5N indicated that each mutant was four and eight times more resistant to sulfonylurea than M342 and PN19, respectively. Protein structure analysis of AHAS1 demonstrated that the leucine of mutant Trp-574-Leu destroyed the original π-plane stacking effect of the local region for tribenuron-methyl binding, leading to herbicide tolerance. Isobole graph analysis showed a significant synergistic effect of the combination of two mutant genes in 5N for improved tolerance to sulfonylurea herbicides. Finally, we bred rapeseed variety Ning R201 using 5N herbicide resistance resources, and observed excellent weed control performance. Together, these results demonstrate the practical value of 5N application for optimizing and simplifying rapeseed cultivation in China.

Variation in mutations providing resistance to acetohydroxyacid synthase inhibitors in Cyperus difformis in China

Cyperus difformis has evolved resistance to pyrazosulfuron-ethyl and other acetohydroxyacid synthase (AHAS) inhibitors in paddy fields in China. To understand the distribution of resistance and the mutations involved, 38 populations collected were from 7 provinces and compared. Application of pyrazosulfuron-ethyl at 30 g a.i. ha−1 identified 16 populations that survived, demonstrating resistance to this herbicide. Two exons of 498 and 1428 bp in length and a 1228–1233-bp intron of AHAS were cloned by genome walking, and three pairs of primers were designed to amplify eight conserved regions in this gene. In the 16 resistant (R) populations, five different types of mutations in the conserved region of the AHAS gene were identified: Pro-197-Ser, Pro-197-Arg, Pro-197-Leu, Asp-376-Glu, and Trp-574-Leu. Three R populations, YX15-22, YX12-10 and YX15-38, were chosen for in vitro AHAS activity assays, and the results showed that AHAS from YX15–22 carrying the Pro-197 mutation was insensitive to pyrazosulfuron-ethyl (resistance index (RI) = 310.0) and penoxsulam (RI = 10.0), whereas the enzyme from YX12–-10 and YX15-38 was insensitive to pyrazosulfuron-ethyl, penoxsulam, imazapic and bispyribac‑sodium (RI values ranging from 4.3 to 4462.0). AHAS inhibitor cross-resistance bioassays showed that YX12-10 and YX15-38 had cross-resistance to all of the tested herbicides (RI values ranging from 5.8 to 3321.9), while the YX15–22 population only had resistance to pyrazosulfuron-ethyl (RI = 827.4) and penoxsulam (RI = 6.6). This study clarified the distribution of resistant C. difformis in China and the different cross-resistance patterns given by various mutation types of AHAS.

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.

Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion.

The acetohydroxyacid synthase (AHAS) is an essential enzyme involved in branched amino acids. Several herbicides wither weeds via inhibiting AHAS activity, and the AHAS mutants show tolerance to these herbicides. However, most AHAS mutations are residue substitutions but not residue deletion. Here, residue deletion was used to engineering the AHAS gene and herbicide-tolerant rice. Molecular docking analysis predicted that the W548 of the AHAS was a residue deletion to generate herbicide tolerance. The AHAS-ΔW548 protein was generated in vitro to remove the W548 residue. Interestingly, the deletion led to the tetramer dissociation of the AHAS, while this dissociation did not reduce the activity of the AHAS. Moreover, the W548 deletion contributed to multi-family herbicides tolerance. Specially, it conferred more tolerance to sulfometuron-methyl and bispyribac-sodium than the W548L substitution. Further analysis revealed that AHAS-ΔW548 had the best performance on the sulfometuron-methyl tolerance compared to the wild-type control. Over-expression of the AHAS-ΔW548 gene into rice led to the tolerance of multiple herbicides in the transgenic line. The T-DNA insertion and the herbicide treatment did not affect the agronomic traits and yields, while more branched-chain amino acids were detected in transgenic rice seeds. Residue deletion of W548 in the AHAS could be a useful strategy for engineering herbicide tolerant rice. The increase of branched-chain amino acids might improve the umami tastes of the rice.

Fluorogenic Assay for Acetohydroxyacid Synthase: Design and Applications.

Acetohydroxyacid synthase (AHAS) exists in plants and many microorganisms (including gut flora) but not in mammals, making it an attractive drug target. Fluorescent-based methods should be practical for high-throughput screening of inhibitors. Herein, we describe the development of the first AHAS fluorogenic assay based on an intramolecular charge transfer (ICT)-based fluorescent probe. The assay is facile, sensitive, and continuous and can be applied toward various AHASs from different species, AHAS mutants, and crude cell lysates. The fluorogenic assay was successfully applied for (1) high-throughput screening of commerical herbicides toward different AHASs for choosing matching herbicides, (2) identification of a Soybean AHAS gene with broad-spectrum herbicide resistance, and (3) identification of selective inhibitors toward intestinal-bacterial AHASs. Among the AHAS inhibitors, an active agent was found for selective inhibition of obesity-associated Ruminococcus torques growth, implying the possibility of AHAS inhibitors for the ultimate goal toward antiobesity therapeutics. The fluorogenic assay opens the door for high-throughput programs in AHAS-related fields, and the design principle might be applied for development of fluorogenic assays of other synthases.

Non-target-site and target-site resistance to AHAS inhibitors in American sloughgrass (Beckmannia syzigachne)

American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) is one of the most competitive and malignant weeds in rice-wheat rotation fields in China. American sloughgrass populations in the Jiangsu Province of China became less sensitive to acetohydroxyacid synthase (AHAS) inhibitors after repeated application for many years in these areas. Two suspected resistant American sloughgrass populations (R1 and R2) collected in the field were detected the resistance to inhibitors of AHAS in whole-plant dose-response assays, compared to the susceptible (S) population. These assays indicated that R1 showed low resistance to mesosulfuron-methyl (3.32-fold), imazapic (2.84-fold) and pyroxsulam (1.55-fold), moderate resistance to flazasulfuron (4.67-fold) and pyribenzoxim (7.41-fold), and high resistance to flucarbazone (11.73-fold). However, using a combination of the cytochrome P450 inhibitor, malathion, with mesosulfuron-methyl resulted in a reduction in R1 resistance relative to mesosulfuron-methyl alone. Furthermore, R2 was highly resistant to flazasulfuron (34.90-fold), imazapic (11.30-fold), flucarbazone (49.20-fold), pyribenzoxim (12.94-fold), moderately resistant to mesosulfuron-methyl (9.77-fold) and pyroxsulam (6.26-fold), and malathion had no effect on R2 resistance to mesosulfuron-methyl. The full-length of AHAS genes was sequenced and the AHAS enzymes were assayed in vitro in order to clarify the mechanism of resistance to AHAS inhibitors in R1 and R2 populations. The results demonstrated that R2 had a Pro-197-Ser mutation in the AHAS gene, and the sensitivity of R2 to the five AHAS inhibitors was decreased, which may result in R2 resistance to AHAS inhibitors. There was no mutation in the AHAS gene of R1, and there were no significant differences in enzyme sensitivity between susceptible (S) and resistant (R1) populations. An enhanced metabolism may be the main mechanism of R1 resistance to AHAS inhibitors.

AHAS Trp574Leu substitution in Raphanus sativus L.: screening, enzyme activity and fitness cost.

Feral radish (Raphanus sativus L.) is a problematic weed that has become resistant to acetohydroxyacid synthase (AHAS) inhibitor herbicides due to the Trp574Leu mutation. An AHAS gene mutation that causes herbicide resistance may have negative pleiotropic effects on plant fitness. This study reports the effects of the Trp574Leu mutation on AHAS activity and reproductive traits of R. sativus. Eight of 17 feral radish accessions presented individuals resistant to metsulfuron-methyl at 0.5% to >90.0% and all the resistant individuals analyzed showed the Trp574Leu mutation. Without herbicide selection, the AHAS activity was 3.2-fold higher in the susceptible accession than in the resistant one. The resistant accession was >9000-fold more resistant to metsulfuron-methyl and imazethapyr than the susceptible accession. Under low intraspecific competition during two growing seasons, AHAS-resistant feral radish accessions showed 22-38% and 21-47% lower seed numbers and yield per plant than the susceptible accession. This is the first report of fitness cost associated with the AHAS Trp574Leu mutation in R. sativus populations. This fitness cost could reduce frequency of the resistant allele without herbicide selection. © 2018 Society of Chemical Industry.

Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene

BackgroundSomatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Solanum tuberosum L.) cultivar ‘Iwa’ using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent.ResultsFollowing 5 days’ exposure of potato cell suspension cultures to 20 μg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7 × 105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase (AHAS) gene. One point mutation involved a transition of cytosine for thymine, which substituted the equivalent of Pro-197 to Ser-197 in the AHAS enzyme. The second point mutation involved a transversion of thymine to adenine, changing the equivalent of Trp-574 to Arg-574. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium-mediated transformation of wild-type ‘Iwa’ potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants.ConclusionsSomatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato.

Tribenuron-methyl resistance and mutation diversity of the AHAS gene in shepherd's purse (Capsella bursa-pastoris (L.) Medik.) in Henan Province, China

Shepherd's purse is a troublesome dicot weed that occurs in the major wheat-producing areas in China. Twenty-eight shepherd's purse populations were collected from winter wheat-planting areas in Henan Province and used to evaluate tribenuron-methyl resistance and acetohydroxyacid synthase (AHAS) gene-mutation diversity. The results indicate that all 28 shepherd's purse populations were resistant to tribenuron-methyl at different levels compared with the susceptible population. Mutation of the 197 codon (CCT) changed proline (Pro) into tyrosine (Tyr), histidine (His), leucine (Leu), serine (Ser), arginine (Arg), alanine (Ala) and threonine (Thr), whereas mutation of the 574 codon (TGG) changed tryptophan (Trp) into leucine (Leu). Among these amino acid changes, a co-concurrence of Pro197Leu and Trp574Leu substitutions was identified for the first time in resistant weed species. Furthermore, Pro197Tyr, Pro197Arg and Pro197Ala substitutions have not been previously reported in shepherd's purse. The results of the in vitro AHAS assay suggest that an insensitive AHAS is likely involved in the resistance to tribenuron-methyl in the R populations with AHAS gene mutations, and the non-target-site based resistance might exist in some populations.

Broad resistance to acetohydroxyacid-synthase-inhibiting herbicides in feral radish (Raphanus sativus L.) populations from Argentina.

Soon after the commercial release of sunflower cultivars resistant to imidazolinone herbicides, several uncontrolled feral radish (Raphanus sativus L.) populations were found in south-eastern Buenos Aires, Argentina. These populations were studied in field, glasshouse and laboratory experiments aiming to characterise their resistance profile and to develop management tools. Three feral radish accessions were highly resistant to ten active ingredients of five families of acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Sequence analysis of the AHAS gene detected a Trp574Leu mutation in all resistant accessions. One accession with an intermediate level of resistance was heterozygous for this mutation, probably owing to gene exchange with a susceptible subpopulation located in the field margin. Herbicide-resistant and herbicide-susceptible radish could be controlled in sunflower by alternative herbicides. This is the first report of feral radish with resistance to herbicides belonging to all the AHAS-inhibiting herbicide families, conferred by Trp574Leu mutation in the AHAS gene. An appropriate herbicide rotation with alternative herbicides such as fluorochloridone or aclonifen and an increase in the diversity of cropping systems are important for minimising the prevalence of these biotypes.

Generation and characterization of tribenuron-methyl herbicide-resistant rapeseed (Brasscia napus) for hybrid seed production using chemically induced male sterility.

Identification and molecular analysis of four tribenuron-methyl resistant mutants in Brassica napus , which would be very useful in hybrid production using a Chemically induced male sterility system. Chemically induced male sterility (CIMS) systems dependent on chemical hybridization agents (CHAs) like tribenuron-methyl (TBM) represent an important approach for practical utilization of heterosis in rapeseed. However, when spraying the female parents with TBM to induce male sterility the male parents must be protected with a shield to avoid injury to the stamens, which would otherwise complicate the seed production protocol and increase the cost of hybrid seed production. Here we report the first proposed application of a herbicide-resistant cultivar in hybrid production, using a CIMS system based on identifying four TBM-resistant mutants in Brassica napus. Genetic analysis indicated that the TBM resistance was controlled by a single dominant nuclear gene. An in vitro enzyme activity assay for acetohydroxyacid synthase (AHAS) suggested that the herbicide resistance is caused by a gain-of-function mutation in a copy of AHAS genes. Comparative sequencing of the mutants and wild type BnaA.AHAS.a coding sequences identified a C-to-T transition at either position 535 or 536 from the translation start site, which resulted in a substitution of proline with serine or leucine at position 197 according to the Arabidopsis thaliana protein sequence. An allele-specific dCAPS marker developed from the C536T variation co-segregated with the herbicide resistance. Transgenic A. thaliana plants expressing BnaA.ahas3.a conferred herbicide resistance, which confirmed that the P197 substitution in BnaA.AHAS.a was responsible for the herbicide resistance. Moreover, the TBM-resistant lines maintain normal male fertility under TBM treatment and can be of practical value in hybrid seed production using CIMS.