Multiple Pro 197 Substitutions in the Acetolactate Synthase of Corn Poppy (Papaver rhoeas) Confer Resistance to Tribenuron Nikolaos S. Kaloumenos, Christos A. Dordas, Grigorios C. Diamantidis, and Ilias G. Eleftherohorinos* 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 (R 1 R 2 ). 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 b-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 Ser, 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. Nomenclature: Tribenuron; corn poppy, Papaver rhoeas L. PAPRH. Key words: ALS-herbicide resistance, point mutations, molecular basis, restriction digestion, protein secondary structure. Acetolactate synthase (ALS; EC 2.2.1.6), also known as acetohydroxyacid synthase (AHAS), catalyses the first com- mon step of the branched-chain amino acid biosynthesis of leucine and valine (condensation of two pyruvate molecules to 2-acetolactate) and isoleucine (condensation of 2-ketobutyrate and pyruvate to 2-acetohydroxybutyrate). These amino acids are essential for plant growth and their absence is lethal for most plant species (Stidham 1991). The number of ALS genes in higher plants is variable. For example, tobacco (Nicotiana tabacum L.) and corn (Zea mays L.) have two unlinked ALS genes (Fang et al. 1992; Keeler et al. 1993), whereas rapeseed (Brassica napus L.) has five ALS genes (Quellet et al. 1992). Corn poppy has only one nuclear ALS gene (Scarabel et al. 2004) as has mouse-ear cress [Arabidopsis thaliana (L.) Heynh] (Mazur et al. 1987). ALS is the target site for the commercially used herbicides of the following five chemical families: sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyloxy(thio)- benzoates, and sulfonylaminocarbonyltriazolinones (Stidham 1991; Tranel and Wright 2002). Crystallographic analysis of this enzyme indicated that the sulfonylurea and imidazolinone herbicides bind to overlapping sites within the substrate access tunnel to the ALS active site and consequently inhibit its activity (Duggleby et al. 2008). ALS-inhibiting herbicides have been widely and rapidly adopted because they combine the advantages of low use rates, low mammalian toxicity, broad-spectrum weed control, and flexible application timing in a wide variety of crops. However, the rapid development of weed biotypes resistant to these herbicides seems to be a major disadvantage that could eventually reduce the usefulness of these herbicides in the near future (Heap 2009; Tranel and Wright 2002). Among the 97 weed species with resistance to these herbicides, the great majority are due to target site mutations of the ALS gene resulting in the change of a single amino acid residue in the catalytic polypeptide chain (Marshall and Moss 2007; Saari et al. 1994; Tranel and Wright 2002). According to Tranel et al. (2009), resistance to ALS-inhibiting herbicides in various plant species is due to six different point mutations that respectively lead to substitution of Ala 122 , Pro 197 , Ala 205 , Asp 376 , Trp 574 , or Ser 653 . Nevertheless, more than half (52%) of the recorded substitutions involve the change of Pro 197 by His, Thr, Arg, Leu, Gln, Ser, Ala, or Ile, while the most frequent recorded substitution was that of Pro 197 by Ser, which represents 28% of the recorded weed R biotypes with Pro substitutions. However, a few resistance cases have been reported due to enhanced rates of herbicide metabolism (Christopher et al. 1991; Preston 2004). Corn poppy populations resistant to sulfonylurea herbi- cides have been found in Italy (Scarabel et al. 2004), Spain (Claude et al. 1998; Dura ´n-Prado et al. 2004), Denmark, and the United Kingdom (Heap 2009). In Greece, a corn poppy population resistant to chlorsulfuron and cross resistant to triasulfuron and thifensulfuron had been reported in 1998 (Heap 2009). However, recent studies showed the presence of corn poppy populations with resistance to tribenuron in more than half of the studied populations originating from winter wheat monoculture located in three different areas in northern Greece (Kaloumenos and Eleftherohorinos 2008). The objective of this study was to sequence the ALS gene from the 28 corn poppy populations with resistance to tribenuron and from 6 S populations in order to identify resistance-conferring mutations and to examine their impact on the predicted protein secondary structure. A rapid and reliable molecular screening method (restriction digestion profile analysis) was also developed in order to detect the existence of point mutations at the codon 197 and discriminate between homozygous and heterozygous R individuals in corn poppy. Materials and Methods Seed Source and Plant Material. Seeds of 28 corn poppy resistant to tribenuron populations along with six S DOI: 10.1614/WS-08-166.1 * First, second, and fourth authors: Laboratory of Agronomy, School of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece; third author: Laboratory of Agricultural Chemistry, School of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece. Corresponding author’s E-mail: eleftero@agro.auth.gr Weed Science 2009 57:362–368 362 N Weed Science 57, July–August 2009