Multiple Pro197 Substitutions in the Acetolactate Synthase of Corn Poppy (Papaver rhoeas) Confer Resistance to Tribenuron

Variations in the acetolactate synthase (ALS) gene sequence were determined from 28 populations of corn poppy resistant (R) to tribenuron and from 6 populations susceptible (S) to this herbicide. The ALS gene fragment (634 bp) sequence revealed in R populations five point mutations at the codon Pro(197), and among them the substitution of Pro(197) by Ala was the most common. The sequencing chromatograms revealed that nine R individuals had only the Mutant ALS gene and were homozygous (RR), 18 R individuals had both the wild type and the mutant ALS gene and were heterozygous (RS), whereas one R individual was heterozygous but with two different mutant ALS alleles (R1R2). The use of restriction digestion profile analysis to verify the DNA sequence results by detecting the existence of point mutations at the codon 197 managed to distinguish the R and S alleles and confirmed the results obtained from the sequencing chromatograms analysis. The secondary protein structure prediction suggested the formation of novel beta-strands for each of the five mentioned amino acid Substitutions that was not present in wild type ALS around the mutant site. These findings support the hypothesis that the substitution of Pro(197) by Set, Thr, Ala, Arg, or Leu resulted in altered secondary structure, which Stabilizes an ALS tertiary conformation that prevents tribenuron binding and thus confers resistance to this herbicide.