Cultivar variation for imazamox resistance in wheat (Triticum aestivum L.): Insights into enzymatic assays for early selection.

Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the target site of several herbicide classes including imidazolinones. Imidazolinone resistance in wheat is conferred by two major genes AhasL-D1 and AhasL-B1. The objective of this work was to evaluate the in vitro and in vivo AHAS activity and plant growth in response to imazamox of nine wheat cultivars. Dose-response curves for two-gene resistant cultivars were significantly different from the single-gene resistant and susceptible cultivars in the in vitro AHAS assay. Resistance levels at the in vivo AHAS and whole-plant assays for resistant cultivars were >10-fold higher than susceptible cultivars. Moreover, in vivo dose-response curves showed differences among cultivars with the same number of resistance genes. It was concluded that in the in vitro AHAS assay cultivar variability was due to differences in target-site sensitivity while the in vivo AHAS assay reflected the resistance at whole-plant level. Both in vitro and in vivo AHAS dose-response curves could be useful tools when exploring mechanisms involved in imidazolinone resistance in different wheat genetic backgrounds and for the selection of higher resistant genotypes.

Effect of Ahasl1-1 and Ahasl1-4 alleles on herbicide resistance and its associated dominance in sunflower.

BACKGROUND: Acetohydroxyacid synthase large subunit 1 (Ahasl1) is a multiallelic locus involved in herbicide resistance in sunflower. Ahasl1-1 and Ahasl1-4 alleles harbor different point mutations that lead to different amino acid substitutions (Ala205Val and Trp574Leu, respectively). The objectives of this work were to evaluate the effect of these alleles at the enzymatic and whole-plant levels, and to determine the dominance relationships for imazapyr and metsulfuron-methyl herbicides. RESULTS: Resistant near-isogenic lines showed significantly lower specific AHAS activity than susceptible near-isoline. However, kinetic studies indicated that mutations did not change AHAS pyruvate affinity. Dose-response for six near-isolines carrying different combinations of Ahasl1-1 and Ahasl1-4 alleles and two herbicides (imazapyr and metsulfuron-methyl) were evaluated at whole-plant and enzymatic levels. Ahasl1-1 allele conferred moderate resistance to imazapyr and low resistance to metsulfuron-methyl. Conversely, Ahasl1-4 allele endowed high levels of resistance for both herbicides. Dominance of resistance at whole-plant level showed a semi-dominant behavior among the alleles for both herbicides. CONCLUSION: Ahasl1-4 allele confers higher resistance levels than Ahasl1-1 when evaluated with imazapyr and metsulfuron-methyl. Dominance estimations suggested that both parental lines should carry a resistance trait when developing hybrids. © 2018 Society of Chemical Industry.

Acetohydroxyacid synthase activity and transcripts profiling reveal tissue-specific regulation of ahas genes in sunflower.

Acetohydroxyacid synthase (AHAS) is the target site of several herbicides and catalyses the first step in the biosynthesis of branched chain amino acid. Three genes coding for AHAS catalytic subunit (ahas1, ahas2 and ahas3) have been reported for sunflower. The aim of this work was to study the expression pattern of ahas genes family and AHAS activity in sunflower (Helianthus annuus L.). Different organs (leaves, hypocotyls, roots, flowers and embryos) were evaluated at several developmental stages. The transcriptional profile was studied through RT-qPCR. The highest expression for ahas1 was shown in leaves, where all the induced and natural gene mutations conferring herbicide resistance were found. The maximal expression of ahas2 and ahas3 occurred in immature flowers and embryos. The highest AHAS activity was found in leaves and immature embryos. Correlation analysis among ahas gene expression and AHAS activity was discussed. Our results show that differences in ahas genes expression are tissue-specific and temporally regulated. Moreover, the conservation of multiple AHAS isoforms in sunflower seems to result from different expression requirements controlled by tissue-specific regulatory mechanisms at different developmental stages.

Precision phenotyping of imidazolinone-induced chlorosis in sunflower.

Chlorosis level is a useful parameter to assess imidazolinone resistance in sunflower (Helianthus annuus L.). The aim of this study was to quantify chlorosis through two different methods in sunflower plantlets treated with imazapyr. The genotypes used in this study were two inbred lines reported to be different in their resistance to imidazolinones. Chlorosis was evaluated by spectrophotometrical quantification of photosynthetic leaf pigments and by a bioinformatics-based color analysis. A protocol for pigment extraction was presented which improved pigment stability. Chlorophyll amount decreased significantly when both genotypes were treated with 10 µM of imazapyr. Leaf color was characterized using Tomato Analyzer(®) color test software. A significant positive correlation between color reduction and chlorophyll concentration was found. It suggests that leaf color measurement could be an accurate method to estimate chlorosis and infer chlorophyll levels in sunflower plants. These results highlight a strong relationship between imidazolinone-induced chlorosis and variations in leaf color and in chlorophyll concentration. Both methods are quantitative, rapid, simple, and reproducible. Thus, they could be useful tools for phenotyping and screening large number of plants when breeding for imidazolinone resistance in this species.

Differential expression of acetohydroxyacid synthase genes in sunflower plantlets and its response to imazapyr herbicide.

Acetohydroxyacid synthase (AHAS) catalyzes the first reaction in branch chain amino acids biosynthesis. This enzyme is the target of several herbicides, including all members of the imidazolinone family. Little is known about the expression of the three acetohydroxyacid synthase genes (ahas1, ahas2 and ahas3) in sunflower. The aim of this work was to evaluate ahas gene expression and AHAS activity in different tissues of sunflower plantlets. Three genotypes differing in imidazolinone resistance were evaluated, two of which carry an herbicide resistant-endowing mutation known as Ahasl1-1 allele. In vivo and in vitro AHAS activity and transcript levels were higher in leaves than in roots. The ahas3 transcript was the less abundant in both tissues. No significant difference was observed between ahas1 and ahas2 transcript levels of the susceptible genotype but a higher ahas1 transcript level was observed in leaves of genotypes carrying Ahasl1-1 allele. Similar transcript levels were found for ahas1 and ahas2 in roots of genotypes carrying Ahasl1-1 allele whereas higher ahas2 abundance was found in the susceptible genotype. Herbicide treatment triggered tissue-specific, gene and genotype-dependent changes in ahas gene expression. AHAS activity was highly inhibited in the susceptible genotype. Differential responses were observed between in vitro and in vivo AHAS inhibition assays. These findings enhance our understanding of AHAS expression in sunflower genotypes differing for herbicide resistance and its response to herbicide treatment.

Soil-less bioassays for early screening for resistance to imazapyr in sunflower (Helianthus annuus L.).

BACKGROUND: Rapid and efficient diagnostic tests for early screening of herbicide resistance are convenient alternatives to field screening methods. There is a need for a quick, reliable and cost-effective method for rapid diagnosis of imidazolinone resistance in sunflower (Helianthus annuus L.). RESULTS: Two seed germination bioassays were developed. Seeds from three sunflower inbred lines differing in resistance to imidazolinones were germinated either on solid culture medium or placed in plastic pots filled with commercial perlite. After 8 days incubation under controlled conditions, both assays successfully distinguished susceptible genotype from the resistant and intermediate ones. The susceptible genotype showed arrested root growth at all herbicide treatments (root length < 1 cm). The resistant genotype developed a complete root system even when exposed to the highest dose of herbicide. However, no definite differences were observed for the intermediate and resistant genotypes with respect to root growth under the different herbicide treatments. CONCLUSION: The simple and rapid screening assays described in the present study were useful in discriminating imidazolinone resistance at the seedling stage. Therefore, these bioassays could be potential tools for early screening of imidazolinone resistance genes from large sunflower populations.