Function of cytochrome P450 CYP72A1182 in metabolic herbicide resistance evolution in Amaranthus palmeri populations

Abstract

Evolution of metabolic herbicide resistance is a major issue for weed management. Few genes and regulatory mechanisms have been identified, particularly in dicotyledonous weed species. We identified putative causal genes and regulatory mechanism for tembotrione resistance in Palmer amaranth (Amaranthus palmeri). Cytochrome P450 candidate genes were identified through RNA-seq analysis. We validated their functions using heterologous expression in Saccharomyces cerevisae. Promoters of the candidate P450 genes were analysed. We performed QTL mapping to identify genomic regions associated with resistance. CYP72A1182 metabolized tembotrione in a heterologous system. The CYP72A1182 gene had increased expression in other A. palmeri populations resistant to multiple herbicides, including tembotrione. Resistant plants exhibited polymorphisms in the promoter of CYP72A1182. We identified quantitative trait loci linked to herbicide resistance, including one on chromosome 4 approximately 3 Mb away from CYP72A1182. CYP72A1182 is likely involved in tembotrione resistance in A. palmeri. Increased expression of this gene could be due to cis-regulation in the promoter, as well as trans-regulation from transcription factors, and further studies are in progress to test this hypothesis. The elucidation of regulatory genes is crucial for developing innovative weed management approaches and target-based novel herbicide molecules.

Keywords: Gene expression regulation; herbicide resistance; hydroxylation; metabolism; monooxygenase; oxidation.

Fig. 1.

Analysis of differentially expressed genes (DEGs) in pseudo-F₂Amaranthus palmeri susceptible (S) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)-resistant (R). (A) Venn diagram of the up- and down-regulated DEGs indicating unique and common DEGs for 18 different comparisons. (B) Heatmap of top DEGs for the comparisons R vs S at 0, 6, and 12 h after herbicide treatment (HAT). (C) Volcano plots displaying gene expression differences between Amaranthus palmeri and treatments of contrast of R versus S before herbicide application (0 HAT), at 6 HAT, and at 12 HAT. DEGs with significant thresholds of P<0.0001, adjusted P<0.05, log2 fold-change >1 or <−1 are represented by red circles. Dashed lines represent significance thresholds of adjusted P<0.05 and log2 fold-change >1 or <−1. (D) Log2 normalized counts of the top four cytochrome P450 genes in response to tembotrione in S and R population, MAKER_25717 (CYP72A1182), MAKER_25718 (CYP72A1027), MAKER_29886 (CYP72A1015), and MAKER_10107 (CYP81CJ2). DEGs had a threshold of adjusted P-value of 0.05 and log2 fold-change 1.

Fig. 2.

LC-MS/MS chromatogram of metabolites formed in yeast transformed with P450 genes from A. palmeri and the Nsf1 gene from maize as a positive control. Blue peak, parental tembotrione; red peak, hydroxy-tembotrione; green peak, dihydroxy-tembotrione. WAT11 (A) and WAT21 (B) yeast strains carrying Arabidopsis cytochrome P450 reductase 1 and 2, respectively. Asterisk indicates at least 10-fold higher peak than the background signal.

Fig. 3.

MUSCLE v5 multiple alignment of CYP81CJ2 (A) and CYP72A1182 (B) promoter sequences. The promoter sequences were reverse-oriented from the reference genome sequence to enable viewing in + strand orientation. Black space indicates differences from the reference Amaranthus palmeri genome (id 55760).

Fig. 4.

Dose–response curve of Amaranthus palmeri herbicide susceptible (NES), resistant (NER), F₁ cross A, and F₁ cross B plants in response to tembotrione.

Fig. 5.

Frequency distribution and genome scan analysis. (A) Distribution of fresh biomass in plants used for QTL mapping of pseudo-F₂ populations from cross A, B, and combined A+B. Blue and red colors represent sensitive and resistant pseudo-F₂ plants, respectively. Dashed line indicates the average biomass for parental NES (blue) and parental NER (red). (B) Genome scan of pseudo-F₂A. palmeri populations from cross A, B, and combined A+B. y-Axis indicates logarithm of the odds (LOD) score statistical estimate, x-axis indicates genome position on scaffolds levels. The plants were treated with 77 g ha⁻¹ of tembotrione, and fresh biomass was measured after 28 d.

Fig. 6.

Markers and resistance phenotypes associated with QTLs for tembotrione resistance in A. palmeri. (A–D) Markers corresponding to the QTL in scaffold 6 (M6). (E–H) Markers corresponding to the QTL in scaffold 10 (M10). (I) Marker in scaffold 14 (M14). Boxplots represent the range of biomass values after herbicide treatment, including the maximum and minimum values, lower and upper quartiles, and the median. Diamonds indicate the average biomass for each genotype.