RESUMO
Protoporphyrinogen oxidase (PPO)-inhibiting herbicides are used to control weeds in a variety of crops. These herbicides inhibit heme and photosynthesis in plants. PPO-inhibiting herbicides are used to control Amaranthus palmeri (Palmer amaranth) especially those with resistance to glyphosate and acetolactate synthase (ALS) inhibiting herbicides. While investigating the basis of high fomesafen-resistance in A. palmeri, we identified a new amino acid substitution of glycine to alanine in the catalytic domain of PPO2 at position 399 (G399A) (numbered according to the protein sequence of A. palmeri). G399 is highly conserved in the PPO protein family across eukaryotic species. Through combined molecular, computational, and biochemical approaches, we established that PPO2 with G399A mutation has reduced affinity for several PPO-inhibiting herbicides, possibly due to steric hindrance induced by the mutation. This is the first report of a PPO2 amino acid substitution at G399 position in a field-selected weed population of A. palmeri. The mutant A. palmeri PPO2 showed high-level in vitro resistance to different PPO inhibitors relative to the wild type. The G399A mutation is very likely to confer resistance to other weed species under selection imposed by the extensive agricultural use of PPO-inhibiting herbicides.
RESUMO
BACKGROUND: Palmer amaranth (Amaranthus palmeri S. Wats) is one of the most common and troublesome weeds in the USA. Palmer amaranth resistance to acetolactate synthase (ALS) inhibitors is widespread in the USA, as in Arkansas. The cross-resistance patterns and mechanism of resistance are not known. Experiments were conducted to determine cross-resistance to ALS inhibitors and identify target-site mutations in 20 Palmer amaranth localities from 13 counties in Arkansas. RESULTS: All Palmer amaranth localities tested had plants cross-resistant to imazethapyr, flumetsulam, primisulfuron, pyrithiobac and trifloxysulfuron. The dose of trifloxysulfuron that caused 50% control was 21-56-fold greater for resistant accessions than for susceptible ones. All but three resistant plants analyzed had one or two relative copies of ALS; one plant had seven relative copies. All resistant plants tested (18 localities) carried the Trp574Leu mutation, which is known to confer broad resistance to ALS inhibitors, supporting the cross-resistance pattern observed. Besides the Trp574Leu mutation, 30% of localities had individuals with one additional resistance-conferring mutation including Ala122Thr, Pro197Ala or Ser653Asn. CONCLUSION: The Trp574Leu mutation in ALS is the primary mechanism of resistance to ALS inhibitors in Palmer amaranth from Arkansas, USA. In some localities, multiple mutations have accumulated in one plant. All localities tested contained plants with resistance to five families of ALS inhibitors. Localities with extremely high resistance to ALS inhibitors, and those outside the subset we studied, may harbor non-target site resistance mechanisms. ALS inhibitors are generally no longer effective on Palmer amaranth in these localities from the US mid-south. © 2018 Society of Chemical Industry.
Assuntos
Acetolactato Sintase/antagonistas & inibidores , Amaranthus/genética , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Acúmulo de Mutações , Proteínas de Plantas/genética , Amaranthus/efeitos dos fármacos , Amaranthus/enzimologia , Arkansas , Proteínas de Plantas/metabolismo , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Plantas Daninhas/genética , Controle de Plantas DaninhasRESUMO
Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in some cases seriously threatens the sustainability of crop production in North America. Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record of glufosinate use at the collection site, survival of plants within the population are likely due to genetic expression that pre-dates selection; in the formal parlance of weed science this is described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was measured using Illumina DNA sequence reads from non-treated and treated surviving (presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282 sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were differentially expressed between treated T and S plants. Of the upregulated genes in treated T plants, 210 were more highly induced than were the upregulated genes in the treated S plants. Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase (GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription factor, NADH-ubiquinone oxidoreductase, ABA 8'-hydroxylase, and cytochrome P450 genes (CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real time-PCR. While GST was upregulated in treated tolerant plants in at least one population, CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are candidates for contributing tolerance to glufosinate. Taken together, these results show that differential induction of stress-protection genes in a population can enable some individuals to survive herbicide application. Elevated expression of detoxification-related genes can get fixed in a population with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained selection pressure could select for mutation(s) in the GS2 gene with the same consequence.