Assessment of ACC and P450 Genes Expression in Wild Oat (Avena ludoviciana) in Different Tissues Under Herbicide Application.

Target-site resistance (TSR) and non-target-site resistance (NTSR) to herbicides in arable weeds are increasing rapidly all over the world and threatening universal food safety. Resistance to herbicides that inhibit ACCase activity has been identified in wild oat. In this study, expression of ACC1, ACC2, CYP71R4 and CYP81B1 genes under herbicide stress conditions were studied in two TSR (resistant in the residue Ile1781-Leu and Ile2041-Asn of ACCase) biotypes, two NTSR biotypes and one susceptible biotype of A. ludoviciana for the first time. Treated and untreated biotypes with ACCase-inhibitor clodinafop propargyl herbicide were sampled from the stem and leaf tissues at 24 h after treatment. Our results showed an increase in gene expression levels in different tissues of both types of resistance biotypes that occurred under herbicide treatment compared with non-herbicide treatment. In all samples, the expression levels of leaf tissue in all studied genes were higher than in stem tissue. The results of ACC gene expression showed that the expression level of ACC1 was significantly higher than that of ACC2. Also, expression levels of TSR biotypes were higher than NTSR biotypes for the ACC1 gene. For both CYP71R4 and CYP81B1 genes, the expression ratio increased significantly in TSR and NTSR biotypes in different tissues after herbicide treatment. In contrast, the expression levels of CYP genes in NTSR biotypes were higher than in TSR biotypes. Our results support the hypothesis that the reaction of plants to herbicide is carried out through a different regulation of genes, which can be the result of the interaction of resistance type in the target or non-target-site.

Resistance determination of the ACCase-inhibiting herbicide of clodinafop propargyl in Avena ludoviciana (Durieu), and study of their interaction using molecular docking and simulation.

Structural mutations providing herbicide resistance may cause a modification of the three dimensional structure of a protein which will lead to a decrease in the herbicide efficacy. Wild oat (Avena ludoviciana Durieu.) is an increasingly disruptive weed in areas of intensive cereal production, thus the aim of this research was to identify mutations conferring resistance to ACCase-inhibitor herbicides at greenhouse, laboratory and in silico scales. Among the selected biotypes, No. 3 in the position 1781 (Ile1781-Leu) and No. 14 in the position 2041 (Ile2041-Asn), showed resistance to ACCase-inhibitor. The above mutations were confirmed using the specific primers and PCR-based methods. Analysis of molecular docking indicated that residues of Trp1948 and Pro2001 are important in the binding site and showed remarkable variation in the mutation types. Using molecular dynamic simulation analysis, we demonstrated that mutation types changed the conformation of the enzyme. These changes resulted in compressed conformation in the active site, which limited the availability of binding herbicide-enzyme. In present, no crystallography molecular structure and modeling reported on the ACCase of plants and this study investigated interactions of clodinafop propargyl and ACCase CT domain in A. ludoviciana by modeling, docking and simulations for the first time. Totally, bioinformatics analysis as well as PCR-based method confirmed that herbicide resistance conferred by nucleotide mutations in the gene sequence.

Validation of expression stability of reference genes in response to herbicide stress in wild oat (Avena ludoviciana).

Weeds are serious problem in crop production and wild oat is a grass weed of economic and agronomic significance. We need to extend our basic knowledge of weeds especially in molecular genetics and gene expression. For study of gene expression by semi-quantitative and quantitative PCR, it is recommended that normalization of reference genes be carried out in order to select the most stable reference gene for a precise gene expression study. The purpose of this research was evaluation of four reference genes in response to treated and untreated (control) by herbicide in two tissues (stem and leaf) of non-target site resistance wild oat (A. ludoviciana). Four candidate reference genes including Actin, Ef1α (elongation factor 1 alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and TBP (TATA-box-binding protein) were used to determine stable reference gene exposed to the herbicide using the statistical methods of NormFinder, BestKeeper and delta-Ct. NormFinder indicated that TBP and Actin genes are the best combination of two genes for normalizing calculations (with a combined gene stability value of 0.012) for qPCR analysis under herbicide stress in different tissues of non-target site resistance wild oat. Based on the statistical results, the Ef1α gene was identified as the unstable reference gene. Totally, according to results of this study, TBP gene is the most stable reference gene and therefore, this gene can be used as a reference gene for future studies of quantitative PCR analysis of herbicide stress-responsive gene expression in wild oat and potentially in other grass weed species.