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Physiological and molecular characterization of mutation-derived imidazolinone resistance in spring wheat.

ACETOHYDROXYACID SYNTHASE, also known as acetolactate synthase (ALS), is a key enzyme in the biosynthesis of the branched chain amino acids valine, leucine, and isoleucine in eukaryotes and prokaryotes (Singh, 1999). The AHAS enzyme from plants has recently been shown to be a tetramer of two catalytic and two regulatory subunits (Lee and Duggleby, 2001, 2002). Five structurally diverse herbicide families, including the sulfonylureas (Ray, 1984), imidazolinones (Shaner et al., 1984), triazolopyrimidines (Subramanian and Gerwick, 1989), pyrimidyloxybenzoates (Subramanian et al., 1990), and sulfonylaminocarbonyl-triazolinones (Santel et al., 1999) are effective in killing susceptible plants by inhibiting AHAS. Plants resistant to one or more of these herbicides have been successfully produced from either seed, microspore, pollen, and callus mutagenesis, or somatic cell selection in maize (Zea mays L.) (Newhouse et al., 1991), Arabidopsis thaliana (L.) Heynh. (Haughn and Somerville, 1986; Sathasivan et al., 1991; Mourad et al., 1993), sugar beet (Beta vulgaris L.) (Hart et al., 1992; Wright and Penner, 1998), canola (Brassica napus L.) (Swanson et al., 1989), cotton (Gossypium hirsutum L.), (Subramanian et al., 1990; Rajasekaran et al., 1996), soybean [Glycine max (L.) Merr.] (Sebastian et al., 1989), tobacco (Nicotiana tabacum L.) (Chaleff and Ray, 1984; Creason and Chaleff, 1988), and wheat (Newhouse et al., 1992; Pozniak and Hucl, 2004). Resistance in most of these cases is due to a form of AHAS that is less sensitive to herbicide inhibition.

In higher plants, point mutations conferring AHAS-inhibitor resistance typically occur in five highly conserved domains in the gene(s) coding for the catalytic subunit of AHAS. Point mutations resulting in proline [(Pro).sub.197] substitutions (amino acid no. based on A. thaliana sequence) are the most commonly reported substitutions, and confer high levels of sulfonylurea resistance with only small increases in resistance to the imidazolinones and the triazolopyrimidines (Haughn et al., 1988; Lee et al., 1988; Guttieri et al., 1992; Harms et al., 1992; Mourad and King 1992; Guttieri et al., 1995; Wright et al., 1998). Alanine [(Ala).sub.205] substitutions in domain D confers moderate resistance to all AHAS inhibitors (Bernasconi et al., 1995; Jander et al., 2003). In domain B, mutations resulting in tryptophan [(Trp).sub.574] substitutions confer high levels of resistance to the imidazolinones, sulfonylureas, and triazolopyrimidines (Lee et al., 1988; Hartnett et al., 1990; Bernasconi et al., 1995; Hattori et al., 1995; Rajasekaran et al., 1996). Point mutations, resulting in [Ala.sub.122] substitutions in domain C, (Wright et al., 1998; Jander et al., 2003) and serine [(Ser).sub.653] substitutions in domain E (Sathasivan et al., 1991; Lee et al., 1999; Jander et al., 2003) confer high resistance solely to the imidazolinones. Although the three dimensional structure of AHAS is not known, a hypothesized structure has been proposed in which the implicated amino acid residues coalesce to form a theoretical herbicide binding site (Ott et al., 1996). No known amino acid substitutions in the regulatory subunit have been reported to confer herbicide resistance.

The imidazolinones are an attractive weed control alternative in common wheat because they are effective at low application rates, control a broad spectrum of weeds, and possess relatively low mammalian toxicity (Warren and Coble, 1999). However, wheat is sensitive to most imidazolinone herbicides (Newhouse et al., 1992; Pozniak and Hucl, 2004). Pozniak and Hucl (2004) were successful in identifying mutant spring wheat plants with moderate to high levels of imidazolinone resistance. The authors identified three independent resistance genes, namely Imi1, Imi2, and Imi3. Newhouse et al. (1992) has shown previously that Imi1 (allelic to FS-4) codes for an altered form of AHAS with reduced imidazolinone sensitivity. The objectives of this study were to determine if Imi2 and Imi3 also code for a herbicide resistant form of AHAS, to sequence the three identified resistance alleles, and to determine the map location of the wild-type genes.


Plant Material

Seeds from homozygous resistant lines TealIMI 10A, TealIMI 11A, TealIMI 15A, BW755 and susceptible CDC Teal (Hughes and Hucl, 1993) were used. Imidazolinone resistant lines TealIMI 10A and BW755 are homozygous for resistance allele Imi1 and TealIMI 11A carries resistance allele Imi2 (Pozniak and Hucl, 2004). TealIMI 15A is homozygous for resistance alleles Imi1 and Imi3 (Pozniak and Hucl, 2004). For the mapping study, nullisomic-tetrasomic and ditelosomic aneuploid fines of T. aestivum 'Chinese Spring' were used (Sears, 1954).

Enzyme Assay for AHAS Activity

AHAS extraction and measurement of AHAS activity was performed on 20-d-old plant material on the basis of procedures modified from Singh et al. (1988). Plants were grown in 16-cm pots (4 plants per pot) containing Rediearth (W.R. Grace and Company, Ajax, ON, Canada) in a walk-in growth chamber with a 16-h-light (22[degrees]C), 8-h-dark (16[degrees]C) cycle. Plants were fertilized with 14-14-14 Nutricote controlled release fertilizer (Chisso-ASAHI Fertilizer Co. Ltd., Tokyo, Japan) after planting. The enzyme was extracted from 3 g of leaf tissue collected from near the coleoptile region. The in vitro assay reactions were performed in 100-[micro]L reactions in 96-well micro-titer plates. Imazethapyr [(RS)-5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid] was diluted such that the final concentrations in the assay reactions were 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 [micro]M. The final concentrations of chlorsulfuron [1-(2-chlorophenylsulfonyl)-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea], a sulfonylurea, in the assay reactions was 0.78 to 100 nM in the same increments used for imazethapyr. Valine and leucine concentrations ranged from 7.81 [micro]M to 1 mM. Acetolactate produced by AHAS was converted to acetoin with 0.30 M [H.sub.2]S[O.sub.4], which was measured by the Westerfeld reaction (Singh et al., 1988). Four reactions without herbicide were included as controls. Background absorbances were measured in reactions with 0.30 M [H.sub.2]S[O.sub.4] added before the addition of the enzyme. Four reactions without enzyme were included as negative controls. Known quantities of bovine serum albumin (BSA) were used to estimate the amount of protein added to each reaction by the Bradford protein assay (Bradford, 1976). The quantity of acetoin produced was estimated on the basis of absorbances of known concentrations of acetoin (3-hydroxy-2-butanone; Sigma Catalogue no. W200816, Sigma, St. Louis, MO) calculated from a standard curve. Specific activities of AHAS from each line were estimated by means of the zero-herbicide control and were calculated as nmoles acetoin [mg.sup.-1] [min.sup.-1]. Four experiments with two replications each were conducted. Absorbance values for each treatment were expressed as AHAS activity (as estimated by absorbance) as a percentage of the mean of the zero-herbicide controls. Data from each line were fit to a nonlinear regression model by PROC NLIN of SAS (SAS Institute Inc, Cary NC, USA). The nonlinear regression was based on a logistic function mathematically described as (Seefeldt et al., 1995)

[1] AHAS Activity (% of the mean of the zero herbicide controls) = [[beta].sub.o] + ([[beta].sub.1] / [[1 + (dose/[I.sub.50]].sup.[[beta].sup.3]]

where [[beta].sub.o] represents the lower asymptote of AHAS activity (%), [[beta].sub.1] represents the mean AHAS activity (%) in the zero-herbicide controls (i.e., upper asymptote), [I.sub.50] represents the dose corresponding to AHAS activity midway between the upper and lower asymptotes (50% response), and [[beta].sup.3] (AHAS activity (%) [dose.sup.-1]) represents the slope of the curve around the [I.sub.50]. Dose represents the concentration of inhibitor used in the enzyme assay. The means of each treatment were estimated by PROC GLM of SAS and plotted on the logistic dose response curves.

Isolation of Genomic DNA, PCR, Cloning

A polymerase chain reaction (PCR) based approach was utilized to identify genes coding for the catalytic subunit peptides of AHAS in common wheat. At Haun stage 2.0 (Haun, 1973), plants were harvested, frozen immediately in liquid nitrogen and stored at -80[degrees]C until DNA extraction. Plants were grown as described for the enzyme assay. DNA extraction was based on the method of Procunier et al. (1990). Primers designed to amplify portions of the catalytic subunit genes from wheat were developed with the nucleotide sequence of five identified contiguous wheat expressed sequence tags (ESTs) (Genbank accession nos. BE429594, BF429146, BE402367, BE402272, BE417248) showing high nucleotide homology with the reported rice (Oryza sativa L.) (GenBank accession no. AB049822) and a partial barley (Hordeum vulgare L.) (GenBank accession no. AF059600) catalytic subunit gene sequences. Primer pair AHAS21FWD (5'-CCGCCGCA ATATGCTATCCAG-3') and AHAS26REV (5'-GTCCTG CCATCACCCTCCATG-3') was designed to PCR clone a 617 bp fragment of the AHAS catalytic subunit genes. This fragment encompasses the [Trp.sub.574] and [Ser.sub.653] residues implicated in herbicide sensitivity in A. thaliana. Primer pair AHAS17FWD (5'-GCCCCGGTCGTCAGGTGTT-3') and AHAS30REV (5- AGTACGAGGTCCTGCCAT CA-3') were designed to PCR clone an 1816-bp fragment of the catalytic subunit genes, including a portion of the putative chloroplast transit peptide, and thus, the DNA sequence coding for the mature catalytic subunits. Polymerase chain reactions were performed in 25-[micro]L reactions consisting of 1 x PCR buffer, 300 [micro]M of each dNTP, 0.5 [micro]M of each primer, and 2.5 Units (U) of Pfu DNA polymerase (Stratagene, La Jolla Ca, USA, Catalogue No. 600136). Inclusion of 5% (v/v) dimethyl sulfoxide (DMSO) in the PCR reaction considerably improved amplification of DNA fragments. The amplicons were separated on either a 1 or 1.5% (w/v) agarose gel containing 0.02 [micro]g m[L.sub.-1] of ethidium bromide (Invitrogen, Burlington, ON, Canada, Catalogue No. 15585011) in 1 x TBE buffer [prepared as a 10 x concentrated stock: 108 g Tris base, 55 g boric acid, 40 mL of 0.5 M EDTA (pH 8.0), volume adjusted to 1 mL]. Following electrophoresis, all fragments of correct length were gel purified with a QIAquick gel extraction kit (Qiagen, Mississauga, ON, Canada, Catalogue No. 28704) and were cloned into the pCR4-Blunt vector with the Zero Blunt Topo Cloning Kit (Invitrogen, Burlington, ON, Canada, Catalogue No. K2875-40) following the instructions of the manufacturer. E. coli cells were plated on selective LB medium 1% (w/v) Tryptone, 0.5% (w/v) Yeast Extract, 1% (w/v) NaCl, 1.5% (w/v) Agar] containing 50 [micro]g m[L.sup.-1] ampicillin (Invitrogen, Burlington, ON, Canada, Catalogue No. 1593019) and grown overnight at 37[degrees]C. Ten to 20 recombinant clones were selected and grown over night at 37[degrees]C in liquid LB medium [1% (w/v) Tryptone, 0.5% (w/v) Yeast Extract, 1% (w/v) NaCl] containing 50 [micro]g m[L.sup.-1] ampicillin. Plasmid DNA was extracted from each of the clones with a Qiaprep Spin Miniprep Kit (Qiagen, Mississauga, ON, Canada, Catalogue No. 2704).

Analysis of Recombinant Clones and DNA Sequencing

Insertion of the PCR fragment into the plasmid vector was confirmed with PCR using PCR primers AHAS21FWD and AHAS26REV and Taq DNA polymerase (Invitrogen, Burlington, ON, Canada, Catalogue No. 18038-018). Following PCR, 5-[micro]L of the reaction were removed and digested with 2.5 Units (U) of MspI (Invitrogen, Burlington, ON, Canada Catalogue no. 15419-013) in a 25-[micro]L reaction at 37[degrees]C for 1 h. MspI is a restriction endonuclease that recognizes and cleaves the 5'-C|CGG-3' sequence. Preliminary experiments showed this enzyme could differentiate cloned wheat catalytic subunit allele sequences into three types on the basis of polymorphic restriction patterns. The digested fragments were separated on a 1.5% (w/v) agarose gel containing 0.02 [micro]g m[L.sup.-1] of ethidium bromide in a 1x TBE working solution. Except for the AhasL-A1 allele from TealIMI 15A, a minimum of four clones (two clones each from two independent PCR reactions) were sequenced. Both strands of inserted DNA fragments were completely sequenced. Only a single clone of AhasL-A1 was isolated from TealIMI 15A. Raw nucleotide sequences were edited by removing the vector sequence and the deduced amino acid sequences were aligned with reported catalytic subunit gene sequences from A. thaliana (imidazolinone resistant line; Genbank accession no. X51514) rice (GenBank accession no. AB049822), and barley (GcnBank accession no. AF059600) by the Megalign program of DNAStar (Lasergene Software, Madison, WI, USA). Only relevant sequence is presented, but all sequences in this manuscript have been submitted to GenBank (; verified 15 March 2004), with the accession numbers being presented.

Chromosome Location

The chromosome locations of wild-type AHAS genes were determined by a PCR-based approach and T. aestivum Chinese Spring aneuploid lines. To map the three genes to specific chromosome arms, PCR was performed on nullisomic-tetrasomic and ditelosomic aneuploid lines representing all 21 T. aestivum chromosomes (Sears, 1954). The primers AHAS21FWD and AHAS26REV were used for successful amplification of a 617-bp amplicon from wild-type Chinese Spring and all aneuploid lines tested. Preliminary results showed that these primers could amplify a 617-bp sequence from all three catalytic subunit genes in a single PCR reaction. Amplicons were digested with MspI as described in the previous section. The three genes could be mapped based on the absence of expected allele-specific MspI digestion patterns in the aneuploid stocks.


Sensitivity of AHAS from Wheat to the Imidazolinones

The presence of AHAS with reduced sensitivity to the imidazolinones was previously demonstrated in T. aestivum cv. Fidel-Imi1 (Newhouse et al., 1992) and it was speculated that resistance genes Imi2 and Imi3 also code for AHAS enzymes with reduced sensitivity to the imidazolinones. In vitro assays of AHAS were conducted on TealIMI 10A (Imi1), TealIMI 11A (Imi2), TealIMI 15A (Imi1 and Imi3), BW755 (Imi1), and CDC Teal (susceptible) to determine if the lines where resistant to imidazolinones and the sulfonylureas at the AHAS level. Specific activities of AHAS in the absence of herbicide ranged from 5.25 [+ or -] 0.57 nmoles acetoin [mg.sup.-1] [min.sup.-1] for BW755 to 5.51 [+ or -] 0.31 nmoles acetoin [mg.sup.-1] [min.sup.-1] for CDC Teal. Since no significant differences in specific activity were detected among the resistant lines and CDC Teal, resistance was not due to an overproduction of AHAS activity.

Resistance in lines tested was due to reduced sensitivity of AHAS to imazethapyr (Fig. 1). The estimates of the lower asymptote of the logistic regression equations (Eq. [1]) represent the mean activities of AHAS at the 100 [micro]M imazethapyr ([[beta].sub.o], Table 1). The AHAS activity from extracts of TealIMI 10A, TealIMI 11A, TealIMI 15A, and BW755 were 40.0 [+ or -] 1.0%, 30.1 [+ or -] 1.6%, 63.1 [+ or -] 3.0%, and 41.4 [+ or -] 2.2% in the presence of 100 [micro]M imazethapyr, respectively, significantly higher (P < 0.05) than the 11.7 [+ or -] 0.8% observed in CDC Teal (Fig. 1; Table 1). Since BW755 and TealIMI 10A both carry Imi1 (Pozniak and Hucl, 2004) both lines were expected to respond similarly to increasing rates of imazethapyr, and confirm the results of Newhouse et al. (1992) that Imi1 codes for an imidazolinone resistant form of AHAS. The activity of AHAS in the presence of imazethapyr was slightly lower in TealIMI 11A when compared with TealIMI 10A and BW755 (Fig. 1). Furthermore, the [I.sub.50] value (dose of imazethapyr corresponding to AHAS activity midway between the upper and lower asymptotes) from TealIMI 11A was 1.8 [micro]M [+ or -] 0.1, significantly less than 2.2 [micro]M [+ or -] 0.1 and 2.3 [micro]M [+ or -] 0.3 observed in TealIMI 10A and BW755, respectively (Table 1). Taken together, these results suggest that the level of in vitro resistance conferred by Imi2 is lower than that observed in the lines carrying Imi1. TealIMI 15A was the most resistant to imazethapyr with 63.1% of the enzyme activity remaining at 100 [micro]M (Fig. 1; Table 1). Furthermore, the [I.sub.50] value for TealIMI 15A was significantly higher than that observed in the other resistant lines evaluated (Table 1).


The AHAS activities in extracts from TealIMI 10A, TealIMI 15A, and BW755 were statistically higher (P < 0.05) than that observed in CDC Teal from 3.13 nM to 100 nM chlorsulfuron (Fig. 2; Table 1 [[beta].sub.o] value), suggesting that AHAS from resistant lines was slightly cross-resistant to the sulfonylurea herbicides. Newhouse et al. (1992) also noted that Imi1 conferred a slight decrease in AHAS sensitivity to the sulfonylureas. As with imazethapyr, the AHAS activities of TealIMI 11A were less than that observed for other resistant lines, and was similar to that in CDC Teal at all concentrations of chlorsulfuron (Fig. 2).


Despite the reduced sensitivity of AHAS to imazethapyr inhibition, AHAS activity from TealIMI 10A, TealIMI 11A, and BW755 displayed in vitro feedback regulation by valine and leucine similar to that in CDC Teal (Fig. 3). The valine + leucine [[beta].sub.o] value from TealIMI 15A was not significantly different from CDC Teal. In this study, the [I.sub.50] value was higher in TealIMI 15A compared to CDC Teal, but this difference was not statistically significant (P > 0.05) (Table 1), suggesting that AHAS sensitivity to valine and leucine were similar between TealIMI 15A and CDC Teal.


Nucleotide Sequence Comparisons between Resistant and Susceptible Lines

In the following, the reference nucleotide sequences coding for the T. aestivum catalytic subunits of AHAS have been submitted to GenBank. Since the complete sequences have not been obtained, all T. aestivum amino acid positions referred to in this manuscript correspond to the rice AHAS catalytic sequence. Two primer pairs (AHAS17FWD-AHAS30REV; AHAS21FWD-AHAS26REV) were used to amplify three partial AHAS catalytic sequences from CDC Teal and resistant lines. Primers AHAS17FWD and AHAS30REV were successful in PCR cloning 1816-bp sequences of imi1 and imi2, whereas Primers AHAS21FWD and AHAS26REV where successful in PCR cloning 617-bp sequences of imi1, imi2, and imi3 from all lines. The three partial sequences could easily be distinguished on the basis of digestion of a 617-bp fragment amplified from cloned sequences with MspI (Fig. 4). Despite the presence of numerous single nucleotide polymorphisms (98.1% identical) between the 1816-bp sequences of imi1 and imi2, only two amino acid differences between the two sequences were noted. Of the 606 amino acids that were sequenced, 93.4 and 93.6% of the amino acids from imi1 and imi2 were conserved with the O. sativa amino acid sequence, respectively. However, only 74.6% of the amino acids in both imi1 and imi2 were conserved with the A. thaliana sequence, suggesting divergence between dicot and monocot catalytic subunit sequences. When comparing the 617-bp partial sequences of imi3 with imi2 and imi3, 99.4% of the nucleotides were identical between imi1 and imi3, whereas 98.1% of the nucleotides were identical when comparing imi2 and imi3.


When comparing TealIM1 10A, TealIMI 15A, and BW755 sequences to CDC Teal, a guanine to adenine transition mutation was noted on the partial Imi1 allele sequence, resulting in a [Ser.sub.627] to [Asp.sub.627] substitution near the carboxyl terminal of the resulting AHAS enzyme (Fig. 5). The same transition mutation was noted on Imi2 of TealIMI 11A (Fig. 5). No other nucleotide mutations where noted on Imi1 and Imi2 (data not shown). To date, we have only been successful in identifying a 617-bp sequence of imi3. On the basis of a single isolated clone from TealIMI 15A, Imi3 does not contain the [Ser.sub.627] substitution noted in Imi1 and Imi2 (Fig. 5). However, since only a single clone was sequenced, these results need to be confirmed with further studies.


Chromosome Location of Ahas Genes

Because of polymorphic MspI restriction digests from each of the ahasL genes (Fig. 4), it was possible to assign each one of the genes to their specific chromosome arms by means of aneuploid stocks of Chinese Spring. The 617-bp sequence from Imi1 lacks an MspI restriction site (Fig. 4) and was absent from nullisomic 6D-tetrasomic 6A (missing 6D chromosomes but possesses four copies of chromosome 6A) (N6DT6A) and ditelosomic 6DS (missing the long arm of chromosome 6D; DT6DS), confirming that Imi1 is located on the long arm of chromosome 6D (Fig. 6). Four MspI fragments were expected from the digestion of Imi2 (Fig. 4). The 63-, 75-, 171-, and 223-bp expected restriction digest pattern was present in wild-type Chinese Spring, but absent in N6BT6D and DT6BS, confirming that Imi2 maps to the long arm of chromosome 6B in T. aestivum (Fig. 6). The N6AT6B and DT6AS lines lacked the 446- and 171-bp fragments resulting from the absence of a single MspI restriction site present on Imi3 (Fig. 6). Therefore, Imi3 was assigned to the long arm of chromosome 6A.


Recommended rules for allele symbolization in T. aestivum suggest "the basic symbol for a gene locus should consist of a two-, three-, or four-letter abbreviation of the trivial name of the enzyme" (McIntosh et al., 1998). Since the resistant genes identified code for the catalytic subunit of AHAS (often referred to as the large subunit of AHAS in the literature), wild-type loci were redesignated as ahasL-D1, ahasL-B1, and ahasL-A1. Since the resistant phenotype is dominant (Pozniak and Hucl, 2004), the resistance alleles were re-designated as AhasL-D1 (previously Imi1), AhasL-B1 (previously Imi2), and AhasL-A1 (previously Imi3).


Results from this study indicate that the imidazolinone resistance phenotype conferred by AhasL-D1, AhasL-B1, and AhasL-A1 is caused by an imidazolinone resistant form of AHAS (Fig. 1). Statistically significant decreases in AHAS sensitivity to the sulfonylureas were also noted at the enzyme level in TealIMI 10A, BW755, and TealIMI 15A (Fig. 2) but not to the same extent noted for imazethapyr (Fig. 1). Furthermore, in field studies, these lines were resistant to the imidazolinones (Pozniak et al., 2004), but extremely sensitive to sulfonylurea herbicides (Pozniak and Hucl, unpublished results). Newhouse et al. (1992) also noted that lines homozygous for AhasL-D1 showed a slight decrease in AHAS sensitivity to the sulfonylureas, but this decrease did not result in whole plant resistance. No decrease in AHAS sensitivity to the sulfonylureas was noted in TealIMI 11A when compared with CDC Teal. Taken together, these results suggest that AhasL-D1, AhasL-B1, and AhasL-A1 provide moderate levels of enzyme resistance to the imidazolinones with little or no resistance to the sulfonylureas. In this study, resistance at the enzyme level was clearly additive, with higher levels of resistance observed in TealIMI 15A, which possesses AhasL-D1 and AhasL-A1. The increased levels of enzyme resistance in TealIMI 15A correlates with higher levels of imidazolinone resistance in the field (Pozniak et al., 2004).

To ensure a balanced supply of the amino acids during plant development, the metabolic regulation of branched chain amino acid biosynthesis involves allosteric regulation of AHAS by the branched chain amino acids (Ouellet et al., 1994). Both valine and leucine have been shown to inhibit AHAS synergistically in plants (Miflin, 1971, Muhitch, 1988; Newhouse et al., 1991; Newhouse et al., 1992). Inhibition of AHAS activity by valine and leucine was similar for the enzyme from wild-type and resistant lines (Fig. 3). These results suggest that, despite increased levels of resistance in TealIMI 10A, TealIMI 11A, TealIMI 15A, and BW755, the synergistic feedback regulation of AHAS by valine and leucine was similar to that seen in CDC Teal (Fig. 3). These results are also consistent with other reports of unaltered AHAS feedback regulation from mutant resistant lines of maize (Newhouse et al., 1991), A. thaliana (Mourad et al., 1995), canola (Hattori et al., 1995), wheat (Newhouse et al., 1992), and tobacco (Creason and Chaleff, 1988).

When comparing AhasL-D1 and AhasL-B1 partial sequences to wild-type ahasL-D1 and ahasL-B1 sequences, a single mutation (guanine to adenine) was noted resulting in a [Ser.sub.627]-[Asn.sub.627] substitution in the resulting AHAS enzymes. The same serine substitution has been implicated in imidazolinone resistance in A. thaliana (Sathasivan et al., 1991; Lee et al., 1999; Jander et al., 2003), rice (Shimizu et al., 2000), and green amaranth (Amaranthus hybridus L.) (Diebold et al., 2002). Results from this study suggest that the [Ser.sub.627]-[Asn.sub.627] substitution also reduces AHAS sensitivity to the sulfonylureas, as noted in TealIMI 10A, BW755, and TealIMI 15A (Fig. 2). These results confirm those of Chang and Duggleby (1998) who also noted that a Ser-Asn substitution resulted in a small reduction in AHAS sensitivity to the sulfonylureas. Bernasconi et al. (1995) noted that the Ser residue implicated in imidazolinone resistance was an Ala residue in imidazolinone susceptible green amaranth. Using site directed mutagenesis, Lee et al. (1999) investigated [Ala.sub.653], [Thr.sub.653], and [Phe.sub.653] substitutions at the [Ser.sub.653] site of the A. thaliana AHAS catalytic subunit and noted that only [Thr.sub.652] and [Phe.sub.653] substitutions resulted in imidazolinone resistance, with the latter also resulting in a slight increase in sulfonylurea resistance. On the basis of these results, Lee et al. (1999) suggested that it is the size of the amino acid side chain at this residue that determines resistance. The side chain of Ala is considerably smaller than that of Ser, whereas the side chains of Asn, Thr, and Phe are considerably larger than that seen in Ser.

In this study, only a single clone of AhasL-A1 was successfully cloned and sequenced from TealIMI 15A and indicated the absence of the [Ser.sub.627]-[Asn.sub.627] substitution noted on AhasL-D1 and AhasL-B1. However, since only a single clone was sequenced, these results are inconclusive and need to be verified. Only three reported amino acids ([Ala.sub.122], [Ala.sub.205] or [Ser.sub.653]) that when substituted result in resistance solely to the imidazolinones. Jander et al. (2003) isolated five A. thaliana mutants with increased resistance to imazethapyr. Of the five, two were [Ser.sub.653] substitutions, and two were [Ala.sub.205] substitutions. A single mutant possessed an [Ala.sub.122] mutation. Since AhasL-A1 does not appear to code for a [Ser.sub.653] substitution, it is possible that the resistance is due to a mutation resulting in either an [Ala.sub.205] or [Ala.sub.122] substitution in the translated catalytic subunit. Studies using genomic DNA libraries derived from CDC Teal are being initiated to identify AhasL-A1 specific PCR primers that will be used to identify the mutation on AhasL-A1 that confers resistance.

Despite AhasL-B1 having the identical base pair mutation as AhasL-D1 (Fig. 5), AhasL-B1 codes for less resistant AHAS activity (Fig. 1). One reason for the lower level of resistance is that AhasL-B1 may be expressed at a lower level than AhasL-D1. In tobacco, Keeler et al. (1993) noted a slightly higher level of transcription of one AHAS gene (SurB) compared with the other (SurA). However, the relative transcription levels of the two genes were consistent in all tissues examined. In addition to two constitutively expressed AHAS genes, canola (Rutledge et al., 1991) and cotton (Grula et al., 1995) were shown to express other AHAS genes in a tissue specific manner. Studies should be conducted to determine the expression level of each of the wheat resistant genes.

The results of the molecular study indicate that common wheat possesses at least three homeologous AHAS catalytic subunit genes, one on each of the three genomes. On the basis of enzyme assay data, AhasL-D1 codes for more resistant activity than AhasL-B1 (Fig. 1). Since TealIMI 15A carries AhasL-D1, the remaining resistant AHAS activity is being coded by AhasL-A1. These data suggest that AhasL-D1 contributes the greatest level of activity to the enzyme pool, with AhasL-A1 contributing the least activity. The three resistance genes additively code for 100% of resistant AHAS activity. Since common wheat is an allohexaploid, at least three homeologous ahasL genes would be expected, one from each of its progenitor species. All three wild-type genes mapped to the long arm of homeologous group six chromosomes (Fig. 6). Similarly, preliminary analysis of EST data from common wheat has indicated the presence of only three copies of ahasL (Ascenzi et al., 2003). In other polyploid species, more than one gene coding for catalytic subunits of AHAS have been characterized. In tobacco, an allotetraploid, two catalytic subunit genes have been identified and characterized, each derived from its ancestral progenitor species N. sylvestris Speg. & Comesand N. tomentosiformis Goodsp. (Mazur et al., 1987). In allotetraploid canola, an AHAS multiallele family consisting of five members (AHAS 1-5) is present (Rutledge et al., 1991), with only two of those genes being constitutively expressed (Ouellet et al., 1992). In allotetraploid cotton, an allele family exists that is composed of six different catalytic subunit coding genes. Two of the AHAS genes were found to be constitutively expressed and are believed to be the main "house-keeping" AHAS genes (Grula et al., 1995). Maize possesses two constitutively expressed genes (Fang et al., 1992).

Seed mutagenesis of common wheat with ethylmethane sulfonate (EMS) (Pozniak and Hucl, 2004) has been effective in generating a single base pair mutation in at least two genes coding for the AHAS catalytic subunit in common wheat. Most EMS induced mutations result in a single base pair GC [right arrow] AT transition, most likely the result of O-6-ethylguanine adducts that mispair with thymine during DNA replication (Snow et al., 1984). Most EMS induced mutations characterized to date occur at a 5'-purine-guanine-3' motif (Bently et al., 2000; Inukai et al., 2000). Both EMS induced mutations identified on AhasL-D1 and AhasL-B1 follow this general pattern. The guanine to adenine substitutions noted on both genes result in GC [right arrow] AT transitions and occur at a 5'-purine (adenine)-guanine-3' motif near the 3' end of the allele (Fig. 5). Jander et al. (2003) isolated 12 EMS induced mutations in A. thaliana resulting in imidazolinone resistance and noted that all identified mutations were single GC [right arrow] AT transitions in the genes coding for the catalytic subunit of AHAS.

The resistance genes characterized in this study will be useful in developing imidazolinone resistant common wheat for commercialization. However, a major concern with developing herbicide resistant wheat is the potential for gene flow to weedy wild relatives. Seefeldt et al. (1998) identified two imidazolinone resistant hybrids derived from the natural hybridization of Aegilops cylindrica Host. (which carries the D genome of common wheat) and an imidazolinone resistant wheat line carrying AhasL-D1 (Newhouse et al., 1992). Results from this study support the hypothesis of Seefeldt et al. (1998) that AhasL-D1 is located on the D genome of common wheat. Wang et al. (2001) suggested that to reduce the risk of gene flow between these two species, breeders developing resistant common wheat should utilize resistance genes located on the A or B genomes. Since the AhasL-B1 and AhasL-A1 loci reside on the long arm of chromosome six of the B and A genomes, respectively, these genes should be preferred by wheat breeders in those areas where imidazolinone resistant common wheat will be commercialized to control A. cylindrica. Furthermore, commercial release of imidazolinone resistant wheat will require producers to intensify weed-resistance management strategies. Imidazolinone-resistant wheat not managed in an integrated weed management system, could contribute to an already significant problem of weed resistance to the imidazolinone herbicides (Heap, 2003).

Results from field studies (Pozniak et al., 2004) suggest that at least two resistance genes are required for adequate levels of imidazolinone resistance in spring wheat. Currently, we are using a combination of genotype analysis, enzyme analysis, and whole plant data to identify the best two-gene combinations that will maximize field resistance to the imidazolinone herbicides.

Abbreviations: AHAS, acetohydroxyacid synthase; N6AT6B; Nullisomic 6A Tetrasomic 6B; N6BT6D, Nullisomic 6B Tetrasomic 6D; N6DT6A, Nullisomic 6D Tetrasomic 6A; DT6AS, Ditelosomic 6AS; DT6AL, Ditelosomic 6AL; DT6BS, Ditelosomic 6BS; DTBL, Ditelosomic 6BL; DT6DS, Ditelosomic 6DS; DT6DL, Ditelosomic 6DL.
Table 1. Nonlinear regression coefficients for AHAS concentration
response curves from four imidazolinone resistant wheat lines and
susceptible cultivar CDC Teal. The nonlinear regression equation is
of the following form: AHAS activity (% of the mean of the zero
herbicide controls) = [[beta].sub.o] + ([[beta].sub.1] -
[[beta].sub.0]) / [1 + [(dose/[I.sub.50]).sup.[[beta]3]]], where
[[beta].sub.o] represents the lower asymptote of AHAS activity (%),
[[beta].sub.1] represents the upper asymptote, [I.sub.50], represents
the dose corresponding to AHAS activity midway between the upper and
lower asymptotes, and [[beta].sub.3] represents the slope of the curve
around [I.sub.50].

Genotype            [[beta].sub.o]       [[beta].sub.1]
                     ([+ or -] SE)       ([+ or -] SE)

  CDC Teal         11.7 [+ or -] 0.8   100.1 [+ or -] 0.7
  TealIMI 10A      40.0 [+ or -] 1.0    99.5 [+ or -] 0.7
  TealIMI 11A      30.1 [+ or -] 1.6    99.9 [+ or -] 0.9
  TealIMI 15A      63.1 [+ or -] 3.0    99.9 [+ or -] 1.4
  BW755            41.4 [+ or -] 2.2   100.2 [+ or -] 1.7
  CDC Teal          7.3 [+ or -] 2.1    99.8 [+ or -] 0.8
  TealIMI 10A      13.3 [+ or -] 1.5    99.8 [+ or -] 0.8
  TealIMI 11A       7.5 [+ or -] 1.4   100.0 [+ or -] 0.6
  TealIMI 15A      12.5 [+ or -] 2.1    99.9 [+ or -] 0.8
  BW755            12.3 [+ or -] 1.5   100.3 [+ or -] 0.7
Valine + leucine
  CDC Teal         39.1 [+ or -] 1.1   100.5 [+ or -] 0.6
  TealIMI 10A      36.6 [+ or -] 2.0    99.7 [+ or -] 0.9
  TealIMI 11A      38.3 [+ or -] 1.0   100.2 [+ or -] 0.6
  TealIMI 15A      38.0 [+ or -] 1.8   100.0 [+ or -] 0.7
  BW755            40.5 [+ or -] 1.5   101.2 [+ or -] 1.0

Genotype              [I.sub.50]        [[beta].sub.3]     [R.sup.2]
                     ([+ or -] SE)       ([+ or -] SE)

  CDC Teal          1.3 [+ or -] 0.1   1.0 [+ or -] 0.1      0.97
  TealIMI 10A       2.2 [+ or -] 0.1   0.9 [+ or -] 0.1      0.98
  TealIMI 11A       1.8 [+ or -] 0.2   0.8 [+ or -] 0.1      0.99
  TealIMI 15A       3.2 [+ or -] 0.9   0.8 [+ or -] 0.2      0.91
  BW755             2.3 [+ or -] 0.3   1.1 [+ or -] 0.2      0.95
  CDC Teal          6.2 [+ or -] 0.4   0.9 [+ or -] 0.1      0.97
  TealIMI 10A       6.7 [+ or -] 0.4   1.1 [+ or -] 0.1      0.99
  TealIMI 11A       6.2 [+ or -] 0.3   0.9 [+ or -] 0.03     0.99
  TealIMI 15A       8.9 [+ or -] 0.6   1.0 [+ or -] 0.05     0.98
  BW755             7.7 [+ or -] 0.4   1.1 [+ or -] 0.1      0.98
Valine + leucine
  CDC Teal         56.8 [+ or -] 3.0   1.2 [+ or -] 0.1      0.98
  TealIMI 10A      60.8 [+ or -] 5.7   1.0 [+ or -] 0.1      0.97
  TealIMI 11A      57.8 [+ or -] 2.8   1.1 [+ or -] 0.1      0.98
  TealIMI 15A      69.3 [+ or -] 6.1   1.0 [+ or -] 0.1      0.97
  BW755            54.3 [+ or -] 4.2   1.2 [+ or -] 0.1      0.96


We are grateful to BASF (formerly American Cyanamid) for providing financial support for this research. The first author also wishes to acknowledge the financial support provided by the Robert P. Knowles Graduate Scholarship.


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Curtis J. Pozniak, * Iwona T. Birk, Louise S. O'Donoughue, Christiane Menard, Pierre J. Hucl, and Bijay K. Singh

C.J. Pozniak and P.J. Hucl, Crop Development Centre, Dep. of Plant Sciences, Univ. of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada, S7N 5A8; I.T. Birk and B.K. Singh, BASF Plant Sciences, Research Triangle Park, Raleigh, NC 27709 USA; L.S. O'Donoughue and C. Menard, DNA LandMarks Inc., 84 Richelieu Street, Saint-Jean-sur-Richelieu, QC, Canada, J3B 6X3. Received 10 Nov. 2003. * Corresponding author (
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Title Annotation:Genomics, Molecular Genetics & Biotechnology
Author:Pozniak, Curtis J.; Birk, Iwona T.; O'Donoughue, Louise S.; Menard, Christiane; Hucl, Pierre J.; Sin
Publication:Crop Science
Date:Jul 1, 2004
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