LIBRARY Mlchigan State University PLACE ll RETURN BOX to remov- thb checkout from your "cord. TO AVOID FINES return on or bdoro date due. DATE DUE DATE DUE DATE DUE mm m1“ WE? MSU In An Affirm-two Action/Equal Opportunity Ira-tumor: Warns-9.1 DEVELOPMENT AND CHARACTERIZATION OF IMIDAZOLINONE-RESISTANT SUGARBEET SOMATIC CELL SELECTIONS By Terry R Wright A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1997 ABSTRACT DEVELOPMENT AND CHARACTERIZATION OF IMIDAZOLINONE-RESISTANT SUGARBEET SOMA'I'IC CELL SELECTIONS By Terry R. Wright Sulfonylureas (SU), imidazolinones (M), and triazolopyrimidine sulfonanilides (TP) are three popular Acetolactate synthase (ALS)-inhibiting herbicide classes commonly used for weed control in crops grown in rotation with sugarbeets. ALS-inhibiting herbicide carryover in soil can severely injure sugarbeets grown in the year(s) following application. Development of sugarbeets resistant to these herbicides may prevent carryover injury , shorten sugarbeet rotation restriction intervals, increase crop rotation flexibility, and provide additional chemical weed control Options for sugarbeets. A sugarbeet (Sur), specifically resistant to SU and TP herbicides was previously generated by somaclonal selection (Saunders et a1. 1992, Crop Sci.32:1357-1360). The utility of this line was limited due to the narrow spectrum of herbicide resistance. Two new herbicide resistant selections, Sir—13 and 9311303, have been isolated with [MI-specific resistance and cross resistance to three classes of ALS-inhibiting herbicides, respectively. Each herbicide resistance was inherited as a monogenic semidominant trait. The herbicide resistance alleles, Sur, Sir-13, and 93R3OB were allelic variants of the single ALS locus in diploid sugarbeets and displayed dominance to the wildtype (wt), herbicide-sensitive allele, but dominance was incomplete among resistance alleles. Sur in vitro shoot cultures were IOOOO-fold resistant to the SU herbicide, chlorsulfuron, and 40-fold resistant to the “FF herbicide, flumetsulam, but not cross resistant to the IMI herbicides. Sur herbicide cross resistance at the whole plant level (>10000-fold to chlorsulfuron postemergence spray applications) and ALS enzyme level (1000- and 50-fold to chlorsulfuron and flumetsulam, respectively) was consistent with shoot culture observations. In contrast, Sir-13 shoot cultures were greater than 100-fold resistant to IMI herbicides in vitro but not cross resistant to SU or TP herbicides. Sir-13 was also more than lOO-fold resistant to soil residues or postemergence applications of imazethapyr and imazamox. ALS enzyme activity from Sir-13 was 40-fold less sensitive to imazethapyr than wildtype, sensitive sugarbeet enzyme, but was not cross resistant to other ALS-inhibiting herbicide classes. 93R30B shoot cultures were 400- to 3600-fold resistant IMI herbicides, >250-fold resistant to IMI herbicides applied postemergence, and >1000-fold resistant at the enzyme level. 93R30B displayed cross resistance to SU and TP herbicides equal to or greater than Sur in shoot culture and ALS enzyme assays. Sir-13 displayed sufficient resistance to avoid carryover injury fi'om field rates of IMI herbicides. Imazethapyr metabolism did not differ among wildtype, Sur, Sir-13, or 93R30B sugarbeets. Deduced amino acid changes in highly conserved ‘herbicide resistance regions’ of the ALS enzyme correlated with the observed cross resistance phenotype in each resistant selection: a serine for prolinem substitution in Sur, a threonine for alanineu3 substitution in Sir-13, and a threonine for alanine"; plus serine for prolinem double mutation in 93R3 OB. 9311303 represents the first plant ALS double mutant derived by a two step selection process which incorporates two class-specific mutations to create a broad cross resistance trait. Dedicated To Wayne Gordon Wright, you have been my inspiration and role model. A quiet man who could move mountains through diplomacy and soft persuasion. Your love and generosity were cherished, your professionalism revered, your integrity respected, your advice trusted, your humor appreciated, and your presence is missed. iv ACKNOWLEDGEMENTS The timely completion of this thesis would not have been possible if not for the support, mentoring, and confidence shown me by Dr. Donald Penner. I sincerely appreciate your advice for my personal as well as professional development. The freedom to pursue research avenues of personal interest is uncommon and genuinely appreciated. The confidence you have shown in me is both humbling and cherished. I am indebted to Dr. Joseph Saunders for the mentoring and patience he showed in teaching this sugarbeet tenderfoot how to grow and breed sugarbeets in the greenhouse. I only wish you could receive the recognition for your contributions to this project that you deserve. For your help and cooperation I am genuinely grateful. I am also sincerely thankful for the time, effort, and interest in this project shown by my other committee members, Drs. Ken Keegstra and Karen Renner. Your individual contributions and suggestions have been helpful and are highly regarded. I am also grateful to Drs. Ken Keegstra and Pam Green for the opportunity to conduct rotations through their laboratories and to learn additional biochemical and molecular techniques from the expertise held in their groups. I also thank Pam Green for helping me to become involved with the MSU-Biotechnology Training Program. The BTP provided an excellent forum to intellectually grow from the collective experiences of the participants. The financial support through my fellowship also allowed me the opportunity to conduct some of my research within an industry setting. The financial support of this project by American Cyanamid and generous use the research facilities in Princeton was greatly appreciated. The genuine interest as well as the moral and technical support of my AmCy colleagues is also sincerely treasured. I am grateful for help and friendship of the entire Plant Biotechnology group but especially thankful for the efforts of Steve Stumer and Dr. Newell Basomb for helping me through the molecular biology portions of this research. I would also like to acknowledge the vigilant support by Drs. Bob Morrison, Laura Sarokin, and Roger Krueger for commercial development of our sugarbeets. Without the hundreds of hours of support given by a wonderful collection of student workers, this project could not have succeeded. I thank Bryan Young, Wendy Pline, Jennifer Rushman, Beth McNeilly, Amy Gonzalez, and Renee Feldpausch for their efforts toward the sugarbeet project. Thanks to Frank Roggenbuck for his moral and technical support and for continually tickling my brain with little “isn’t this cool” observations and discussions. The fellowship of the graduate students and postdocs of the PRL and Weed Science group is also appreciated. I must acknowledge my parents, Wayne and Shirley, for their wonderfirl support and encouragement through 23 years of school. You were my first teachers and my most cherished fiiends. Finally, my heartfelt thanks go to my wife, Theresa, for your tireless support, fi'iendship, and love through all that I have put you through. Thank you for your understanding and sacrifice. I am blessed beyond measure to have your support and vi commitment to my success. The completion of this work is a measure of your dedication as much as mine. Thank you. vii TABLE OF CONTENTS LIST OF TABLES ................................................................................ LIST OF FIGURES .............................................................................. CHAPTER 1 Literature review .................................................................................. Introduction .............................................................................. ALS-inhibiting herbicide mode of action ............................................. ALS enzyme characteristics ............................................................ Herbicide inhibition of ALS ............................................................ ALS-inhibiting herbicide selectivity ................................................... Importance of sugarbeet ................................................................ Sugarbeet weed control ................................................................. Crops resistant to ALS-inhibiting herbicides ......................................... Weed resistance to ALS-inhibiting herbicides ........................................ Imparting ALS-inhibiting herbicide resistance to crops ............................. ALS mutations associated with herbicide resistance ................................ Statement of the problem and attribution ............................................. Literature cited ........................................................................... CHAPTER 2 Selection and inheritance of imidazolinone resistance in sugarbeet (Beta vulgaris). . . .. viii xii xiv l 2 3 4 8 10 12 13 15 l7 19 24 35 39 107 Abstract ................................................................................... 108 Introduction .............................................................................. 109 Materials and Methods .................................................................. 111 Somatic cell selection ........................................................... 111 Tissue culture media ................................................... 111 Imidazolinone resistance .............................................. 112 Imidazolinone and sulfonylurea resistance .......................... 114 Genetic analysis ................................................................. 114 Imidazolinone resistance .............................................. 114 Imidazolinone and sulfonylurea resistance .......................... 115 Allelism of herbicide resistance traits ......................................... 116 Genetic evidence ....................................................... 116 Southern blot ananysis ................................................ 117 Results and discussion .................................................................. 119 Somatic cell selection ........................................................... 119 Imidazolinone resistance .............................................. 119 Imidazolinone and sulfonylurea resistance .......................... 119 Genetic analysis ................................................................. 120 Imidazolinone resistance .............................................. 120 Imidazolinone and sulfonylurea resistance .......................... 121 Allelism of herbicide resistance traits ......................................... 122 Genetic evidence ....................................................... 122 Southern blot analysis ................................................ 124 Acknowledgements ...................................................................... 125 Literature cited ........................................................................... 126 CHAPTER 3 .1" Wire and whole plant magnitude and cross resistance characterization of two unidazolinone-resistant sugarbeet (Beta vulgaris) somatic cell selections ................. 139 Abstract ................................................................................... 140 Introduction .............................................................................. 141 Materials and Methods .................................................................. 144 In vitro shoot culture resistance .............................................. 144 Whole plant resistance to simulated imidazolinone herbicide soil residues ........................................................................... 145 Whole plant resistance to POST herbicide applications .................... 146 Sir-13 allele copy number effect on POST imidazolinone resistance ..... 147 ix Results and discussion ................................................................... 148 In vitro shoot culture resistance .............................................. 148 Whole plant resistance to simulated imidazolinone herbicide soil residues ........................................................................... 150 Whole plant resistance to POST herbicide applications .................... 151 Sir-13 allele copy number effect on POST imidazolinone resistance. . . .. 153 Acknowledgements ...................................................................... 153 Literature cited ........................................................................... 155 CHAPTER 4 164 Biochemical mechanism and molecular basis for ALS-inhibiting herbicide resistance in sugarbeet (Beta vulgaris) somatic cell selections .............................. Abstract ................................................................................... 165 Introduction .............................................................................. 167 Materials and Methods .................................................................. 169 l4C-imazethapyr metabolism .................................................. 169 ALS enzyme assays ............................................................. 172 ALS gene sequencing ........................................................... 174 Automated sequencing ................................................ 174 Manual sequencing .................................................... 177 Results and discussion ................................................................... 178 l‘C-imazethapyr metabolism .................................................. 178 ALS enzyme assays ............................................................. 179 ALS gene sequencing ........................................................... 183 Automated sequencing ................................................ 183 Manual sequencing .................................................... 183 Acknowledgements ...................................................................... 188 Literature cited ........................................................................... 189 CHAPTER 5 Conclusions ........................................................................................ 212 Positive implications of imidazolinone-resistant sugarbeets ........................ 213 Negative implications of imidazolinone-resistant sugarbeets ....................... 216 Literature cited ........................................................................... 218 APPENDIX A ALS—inhibiting herbicide chemical names ...................................................... 220 APPENDIX B One- and three-letter abbreviations for amino acids .......................................... 225 LIST OF TABLES CHAPTER 1 Table 1 Currently marketed and experimental ALS-inhibiting herbicides... 62 Table 2 Rotation restrictions for sugarbeets with ALS-inhibiting herbicides .................................................................. 65 Table 3 Weeds resistant to ALS-inhibiting herbicides isolated fi'om 67 natural populations ........................................................ Table 4 Laboratory derived plants resistant to ALS-inhibiting herbicides... 70 Table 5 Unique selected or engineered ALS amino acid substitutions associated with herbicide resistance in various species ............... 77 Table 6 Examples of different amino acid residue numbers identifying firnctionally equivalent amino acids (FEAA) with respect to ALS-inhibiting herbicide resistance ..................................... 82 Table 7 Herbicide cross resistance phenotype summary for amino acid substitutions fi'om the wildtype firnctional equivalent amino acid (FEAA) for plants, yeast, and bacteria ................................. 85 CHAPTER 2 Table 1 Phenotype of sugarbeet ALS alleles .................................... 131 Table 2 IMI-R genotype distribution for Sir-13 F2 progeny test .............. 132 Table 3 IMI-R/SU-R phenotype distribution for 93R3OB F2 herbicide resistance screen ................................................ 133 Table 4 Chi square analysis of observed 93R3OB F2 progeny 134 test phenotype distribution versus proposed one or two ALS loci models ........................................................... CHAPTER 3 Table 1 Shoot culture magnitude and cross resistance to various 160 ALS-inhibiting herbicides. : ............................................... xii Table 2 Table 3 Table 4 CHAPTER 4 Table 1 Response of Sir-13/Sir-I3 F3 plants to simulated (PPI) 161 imidazolinone carryover residues ..................................... Whole plant sugarbeet response to POST-applied IMI and SU herbicides ................................................... 162 Effect of Sir-13 allele copy number on resistance to POST 163 imidazolinone applications ............................................... ALS enzyme response to three classes of inhibitors .................. 199 xiii CHAPTER] Figure 1 Figure 2 Figure 3 Figure 4 CHAPTER 2 Figure 1 Figure 2 CHAPTER 4 Figure 1 Figure 2 Figure 3 LIST OF FIGURES Representative compounds from four commercialized ALS-inhibiting herbicide classes ........................................ 86 Biosynthetic pathway for the branched-chain amino acids .......... 88 Deduced amino acid sequence alignments of ALS enzymes from eight plant species, yeast, and E. coli .............................. 90 Model ALS enzyme primary protein structure indicating relative positions of conserved amino acid residues implicated in resistance to ALS-inhibiting herbicides when changed from the wildtype amino acid corresponding to the FEAA residues in Figure 3 ........................................................ 105 Phenotype evaluation to determine herbicide resistance allelism. .. 135 Sugarbeet Southern blot analysis for ALS gene copy detemrination .............................................................. 137 Deduced amino acid substitutions within the ALS enzyme primary structure of selected plant species resulting in resistance to one or more classes of ALS-inhibiting herbicides ..................... 200 Wildtype (wt) sugarbeet ALS nucleotide and deduced amino acid sequences (nucleotide positions 301-660; amino acid positions 101-220) surrounding and including Region A (shaded) from Figure 1 .................................................... 202 Wildtype (wt) sugarbeet ALS nucleotide and deduced amino acid sequences (nucleotide positions 1621-2027; amino acid positions 541-665) surrounding and including Region A (shaded) fiom Figure 1 .................................................... 204 xiv Figure 4 Figure 5 Figure 6 Metabolism of parent [l4C]-imazethapyr and accumulation of the a-hydroxyethyl-imazethapyr metabolite in four sugarbeet lines .............................................................. 206 Sequencing gel of sugarbeet ALS Region A showing two 208 nucleotides where resistant lines showed one or more changes fi'om the wildtype (wt) sequence ......................................... Deduced amino acid composition for three ALS-resistant sugarbeet selections and a sensitive wildtype (wt) sugarbeet at the five known plant ALS sites for herbicide resistance ......... 210 Chapter 1 LITERATURE REVIEW 2 Introduction. The introduction of new varieties and increased mechanization, application of inorganic fertilizers, and the incorporation of effective crop protection chemicals have impacted cropping efficiency and yields in a synergistic manner. World average corn (Zea mays L.), wheat (T riticum aestivum L.), and rice (Oryza sativa L.) yields have increased three to five fold in the past 100 years because of the new technologies introduced into crop production (Chrispeels and Savada 1994). The use of synthetic chemicals for weed control has steadily grown in the post-World War II era following the commercial introduction of 2,4-D (Gebhart and McWhorter 1987). Herbicides have gained popularity because they can effectively and selectively remove weeds from a crop, saving labor and resource costs for hand-removal or mechanized tillage. Some herbicides also have long- lasting effects, controlling late-germinating weeds if residual herbicide concentrations remain high enough in the soil. The herbicide market has reached maturity in the United States, with nearly 96% of com, 97% of soybean [Glycine max (L.) Merr.], 90% of cotton (Gosnpium hirsumm L.), and 51% ofwheat acres receiving at least one herbicide (Lin et a1. 1995). The herbicide market continues to grow in Third World countries; however, recent focus in industrialized nations has been to reduce the chemical load on the GHVironment and discover efi‘ective chemicals with improved environmental and t(”Kicological properties. In this effort, herbicide technology made a tremendous leap in 1982 with the introduction of the herbicide chlorsulfiiron for use in cereals (Saari et a1. 1994). Chlorsulfuron is a member of the sulfonylurea (SU)l class of herbicides. SU herbicides ‘ Abbreviations; SU, sulfonylurea; ALS, acetolactate synthase (EC. 4.1.3.18, also known as AHAS, acmohYdroxyacid synthase); IMI, imidazolinone; TP, triazolopyrimidine sulfonanilide; PTB, (Ma inirc corm 111311 Prim each area 3 became extremely popular due to their high efiicacy, low toxicity, and low use rates (Mazur and Falco 1989). These herbicides are effective at low rates related to their highly specific inhibition of the enzyme acetolactate synthase (ALS)l (Ray 1984). Since the introduction of SU herbicides, three new classes of herbicide chemistry have been commercialized that also inhibit ALS: the irnidazolinones(Il\41)l (Shaner et al. 1984), the triazolopyrimidine sulfonanilides (TP)l (Gerwick et al. 1990), and the pyrimidinylthiobenzoates (P'I'B)1 (Takahashi et al. 1991). Structural representatives for each herbicide class are shown in Figure 1. Table 1 lists 36 ALS-inhibiting herbicides that are currently marketed or are nearing commercial release (See Appendix A for chemical names of herbicides). Other chemistry classes do inhibit ALS activity and are under investigation for potential herbicide development (Babczinski and Zelinski 1991). ALS-inhibiting herbicide mode of action. Early work with the experimental SU and M herbicides identified the branched-chain amino acid pathway as the target site for these herbicides. Several lines of evidence support this observation. Treatment of corn cell cultures with irnazapyr reduced levels of fiee valine, leucine, and isoleucine in the Cilltures versus untreated controls (Anderson and Hibberd 1985). The grth reduction Effects of imazapyr were reversed by supplementing cultures with exogenous leucine and Pyrminidinylthiobenzoate; POB, pyrimidinyloxybenzoate; FAD, flavin adenine dinucleotide; TPP, thiamine pyrophosphate; HETPP, hydroxyethyl thiamine pyrophosophate; kD, kilodaltons; 1,0, Concentration required to reduce enzyme activity 50%; BA, bioassay; SU-R, sulfonylurea resistant; IMI-R, imidazolmonc resistant; SU-R/IMI-R, broad cross resistant; EMS, ethyl methane sulfonate; NMU, N- nitI'Oso-N-rnethyl urea; FEAA, functional equivalent amino acid; OTP, organellar transit peptide; POX, Pymvare oxidase (EC 1.2.3.3). 4 valine. Imazapyr injury symptoms were alleviated by supplementing sprayed plants with branched-chain amino acids (Shaner and Reider 1986). The penultimate intermediates to isoleucine and valine synthesis were also able to reverse chlorsulfuron effects on pea root cultures, indicating SU herbicides indeed directly afl‘ected the branched-chain amino acid pathway (Ray 1984). Finally, ALS-inhibiting herbicide-resistant isolates of corn (Anderson and Georgeson 1989; Bright et al. 1992), tobacco (Nicotiana tabacum L.) (Chalefi‘ and Ray 1984; Creason and Chalefi‘ 1988) and many other species have been isolated. In nearly all cases, whole plant resistance has strictly segregated with a herbicide-insensitive form of the ALS enzyme. Isolation of genes coding for a resistant ALS enzyme and transformation of a sensitive plant with the resistance gene has produced a similar resistance phenotype in the transformed plant (Hng et al. 1988; McHughen 1989; D’Halluin et al. 1992). These experiments clearly identified the branched chain amino acid biosynthetic pathway (and specifically ALS) as the site of action of the herbicides identified as “ALS-inhibiting herbicides” (Table 1). ALS enzyme characteristics. ALS catalyzes the first reaction common to the biosynthesis of all branched-chain amino acids (valine, leucine, and isolecuine). ALS requires three cofactors for fiill enzyme activity: flavin adenine dinucleotide (F AD)‘, thiamine pyrophosphate (TPP) ‘, and Mg+2 (Singh et al. 1988a). ALS catalyzes a non- oxidative decarboxylation of pyruvate via a hydroxyethyl thiamine pyrophosphate (I'IETPP)l intermediate with subsequent condensation with a second pyruvate molecule or 2-ketobutyrate to form acetolactate or acetohydroxybutyrate, respectively, and concomitant release of one CO; molecule (Hawkes et al. 1989; Stidham and Singh 1991). 5 FAD is believed to play an enzyme-stabilizing role in ALS and is not involved in electron transfer common with many oxidative fiavoenzymes (Schloss et al. 1988; Muhitch et al. 1987; Singh and Schmitt 1989; Burner and BOger 1991). Parallel pathways for the synthesis of isoleucine or valine and leucine, the branched- chain amino acid end products, are catalyzed by the same bifunctional enzymes (acetohydroxyacid reductoisomerase and dihydroxyacid dehydratase) in the next two steps (Umbarger 1978; Stidham and Singh 1991). Valine and isoleucine are formed by transarnination of 2-keto-isovalerate and 2-keto-methylvalerate, respectively, catalyzed by valine arninotransferase. Leucine synthesis branches from the common pathway, utilizing 2-keto-isovalerate and four enzymes (2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isoproprylmalate dehydrogenase, and leucine arninotransferase) (Stidham 1991) (Figure 2). Plants and microbes alike utilize the same pathway to produce needed branched chain amino acids. Much of the early work on the biochemistry, regulation, and enzymology of this pathway was conducted with bacterial systems. While many aspects of this pathway are the same or closely similar between plants and microbes, there are also distinct difi‘erences. For this reason, microbial investigations and results have been viewed only as models from which to design plant paradigms for the same pathway. Microbial control of the pathway includes feedback inhibition by pathway endproducts as well as difi‘erential expression of the three microbial ALS isozymes regulated by the amino acid endproducts or a catabolite repression system (Umbarger 1978; Barak et al. 1987, 1988, 1990; Gollop et al. 1989, 1990). Pathway regulation in plants appears to rely specifically on feedback inhibition mechanisms. In plants, threonine dehydratase is inhibited by isoleucine (Shanna and Mazumder 1970), 2-isopr0pylmalate synthase by 6 leucine (Oaks 1965), and ALS by all three end products (Miflin 1971; Miflin and Cave 1972; Singh et al. 1988a). ALS isozymes have been purified to homogeneity in Escherichia coli (Grimminger and Umbarger 1979; Eoyang and Silverrnan 1984; Barak et al. 1988) and Salmonella whimurt'um (Schloss et al. 1985). In all cases, the enzyme was found to be a heterotetramer composed of two identical large and small subunits. Catalytic activity of ALS was ascribed to the large subunit and the small subunit appeared to be responsible for feedback inhibition by valine, leucine, and isoleucine (Eoyang and Silverrnan 1986; Weinstock et a1. 1992). Rigorous enzyme studies with plant ALS are difficult because enzyme extracts are extremely labile (Shaner et al. 1984; Muhitch et al. 1987). An ALS holoenzyrne has not been purified to homogeneity and the subunit composition of plant ALS remains unclear. However, ALS genes have been isolated fi'om several plant species (Mazur et al. 1987; Lee et a1. 1988; Bekkaoui et al. 1991; Rutledge et al. 1991; Fang et al. 1992; Grula et al. 1995). The deduced amino acid sequences from these genes display a high degree of homology to the bacterial ALS large subunit (Mazur and Falco 1989; Hartnett et al. 1991; Bedbrook et al. 1995). To date, a small subunit homologue has not been identified, although circumstantial evidence suggests an additional ALS subunit with allosteric effecting properties may also be associated with plant ALS enzymes (Singh et a1. 1992). Several groups have semi-purified multiple forms of ALS from plants difi‘ering in size, feedback and herbicide sensitivity, and degree of oligomerization. Singh et al. (1988b) separated two forms of ALS (AHAS I and AHAS II) from corn suspension cultures representing 90 and 10% of the extracted ALS activity, respectively. AHAS I had an estimated molecular weight of 193 kilodaltons (kD) 1; a somewhat broad pH optimum of 6 7 to 7; K. for pyruvate of 5 mM; 150‘ (the concentration required to reduce enzyme activity 50%) values for feedback inhibitors (leucine + valine), imazapyr, and sulfometuron of 0.1 mM, 2.0 nM, and 10.0 nM, respectively. On the other hand, AHAS II had a relative molecular weight of 55 kD, a sharp pH optimum of 7, slightly higher K... for pyruvate (8 mM); was inhibited 10% by 1 mM leucine + valine; was slightly less sensitive to imazapyr (Iso=1.5 Md); and possessed the same sensitivity to sulfometuron (Singh et al. 1988b). The authors suggested this may represent oligomeric (AHAS I) and monomeric (AHAS II) forms of ALS or different isozymes of ALS. The former hypothesis was supported by fiirther studies with corn cell ALS showing FAD caused increased oligomerization of ALS extracts fi'om the dimer (150 kD) to tetramer (300 kD) (Singh and Schmitt 1989). The tetramer form was more sensitive to feedback inhibition as well as inhibition by imazapyr. The authors suggested that FAD played an important structural role in ALS subunit association. Similar observations have been made in com (Muhitch et al. 1987), and barley (Hordeum vulgare L.) (Dumer and BOger 1991). Other factors also affect the degree of subunit aggregation. Fresh corn ALS extracts were compared with extracts aged 4 weeks at -20 C for degree of oligomerization and sensitivity to feedback inhibitors and imazapyr. Fresh extracts predominated in the dimer and tetramer forms which displayed a sensitivity to feedback inhibitors (leucine + valine) and imazapyr; aged extracts were predominantly in the monomeric form and were highly resistant to feedback inhibition although inhibition by imazapyr was unchanged (Stidham 1991). The lability of feedback inhibitor sensitivity was also affected by temperature. As incubation temperature increased from 37 to 50 C, the level of inhibition by valine + leucine (1 mM each) decreased from 74% to 26% (Stidham 1991). The sensitivity to 8 imazapyr, however, was constant across this temperature range. The lability of the dimer and tetramer ALS forms considered with the lability of feedback inhibitor sensitivity in aged or heated extracts would support the hypothesis that at least some degree of oligomerization occurs in viva. Other studies support homo-dimer, -tetramer, or higher level of aggregation of ALS subunits in other species (Muhitch et al. 1987; Dumer and BOger 1991; Bekkaoui et al. 1993). The exact composition of the high molecular weight aggregate, however, has not been determined and leaves room for speculation that a homologous small subunit may exist in plants. Overexpression of Arabidopsis thaliana (L.) Heyhn. ALS in E. coli (PAIO) yielded a mature enzyme of similar size (65 kD) to denatured extracts (monomer) fi'om Arabidopsis plants with similar sensitivity to imazethapyr and chlorsulfuron as seen with fi'esh plant extracts (Singh et al. 1992). Arabidopsis ALS expressed in E. coli in either the monomer or dimer form, however, was insensitive to valine + leucine (1 mM each) differing greatly from the plant extracts that displayed 60% inhibition by valine + leucine. Since only the large subunit homologue was expressed in E. coli, the authors suggested a plant ALS small subunit may exist which imparts feedback inhibitor sensitivity to the holoenzyme. This subject remains an area of active investigation. Herbicide inhibition of ALS. The effect of many herbicides on in vitro ALS activity has been examined. Consistently, the herbicides in Table 1 have been identified as potent inhibitors of ALS activity. A few detailed enzymatic analyses have attempted to determine the mechanism of inhibition of ALS activity, often with conflicting results. SU herbicides exhibit mixed competitive or noncompetitive ALS inhibition with respect to pyruvate (Hawkes et al. 1989; Schloss 1990; Dumer et al. 1991; Gerbling and K6tter 1991). IMI 9 herbicides have been classified as noncompetitive or uncompetitive ALS inhibitors with respect to pyruvate (Shaner et al. 1984; Dumer et al. 1991; Gerbling and KOtter 1991; Hawkes et al. 1992). The TP herbicides appear to be noncompetitive inhibitors with respect to pyruvate (Hawkes et al. 1989; Gerbling and Katter 1991). PTB herbicides were competitive or uncompetitive with respect to pyruvate depending on the length of incubation with the inhibitor (Gerbling and KOtter 1991). Most studies indicate there is a bi-phasic type of inhibition displayed with each of these classes of herbicides. The inhibition constant decreases fiom its initial value to a final inhibition constant that is 10- to 20-fold lower (greater inhibitor afinity for the enzyme). This type of inhibition phenomenon occurs with slow, tight-binding inhibitors and indicates there may be a shift within the ALS active site after initial binding of the herbicides to a more stable, tight- binding conformation (Hawkes et al. 1989; Shaner et al. 1990; Schloss 1990). In viva evidence of a tight-binding inhibition mechanism of inhibition was shown by the 90% reduction of extractable ALS activity in corn shoots treated with imazapyr 4 h before enzyme extraction (Stidham and Shaner 1990), indicating in viva ALS-bound imazapyr was not displaced during the extraction procedure. Further evidence for tight binding was shown by recovery of ["C]-imazapyr in the ALS fi'action of extracts from corn leaves pretreated with radiolabeled imazapyr (Shaner et al 1990). The recovered [“C]-imazapyr present in the enzyme assays (contributed from enzyme extracts from treated plants) without additional herbicide added was determined to be about 50 nM. The ALS activity in these extracts was inhibited 75% versus enzyme activity of herbicide- fi'ee assays of enzyme extracts fiom non-treated corn plants (Shaner et al. 1990). The imazapyr concentration required to inhibit 50% of enzyme activity fiom nontreated plants 10 was about 100 times higher than the 50 nM imazapyr extracted with the ALS enzyme from treated plants. These results suggest a highly specific and tight binding association of imazapyr for the ALS enzyme. ALS-inhibiting herbicide selectivity. Animals lack the ALS enzyme, thus resulting in a high margin of safety between target plants and nontarget animals. Among plants, however, ALS-inhibiting herbicide selectivity is generally afl‘orded by faster herbicide metabolism in the tolerant versus sensitive species (Brown et al. 1990; Hartnett et al. 1991; Shaner and Mallipudi 1991; Lee et al. 1991; Tecle et al. 1993). Enzyme sensitivity to ALS-inhibiting herbicides does vary among plant species, but whole plant sensitivity usually does not correlate with inherent enzyme sensitivity (Stidham and Singh 1991). Instead, plant tolerance correlates with the rate and extent of herbicide metabolism. ALS-inhibiting herbicides control a broad spectrum of weeds in corn, leguminous and small grains crops, as well as industrial and noncrop sites. Most of these chemicals are applied POST, but some are soil-applied as PPI or PRE treatments. Late-germinating weeds are controlled by the high activity of these compounds and the ability of plants to take up the chemical from the soil in high enough concentrations to be phytotoxic. Unfortunately, the residual activity of ALS-inhibiting herbicides can also injure susceptible rotational crops (Renner and Powell 1990; Goetz et al. 1990; Walsh et al. 1993; Johnson et al. 1993; Krausz et al. 1994; Kotoula-Skya et al. 1993). Sugarbeets (Beta vulgaris L.) and lentils (Lens esculenta L.) are more sensitive to ALS-inhibiting herbicide residues than most other crops (Moyer et al. 1990; Kotoula-Skya 1993). Label restrictions prevent sugarbeets from being planted in fields where ALS-inhibiting herbicides have been applied for intervals ranging from 2 to 40 months (Table 2). The potential for herbicide carryover l 1 injury can be firrther complicated by soil and environmental factors which afl‘ect the rate and extent of herbicide dissipation (F rederickson and Shea 1986; Stougaard et al. 1990; Brown 1990). Soil pH plays an important role in SU and IMI herbicide persistence. Interestingly, the persistence of each herbicide class is affected in an opposite manner by soil pH. The “sulfonylurea bridge” connecting the two ring structures of SU compounds is susceptible to acid hydrolysis (Fuesler and Hanafey 1990; Schneiders et al. 1993 ) and is therefore more susceptible to soil degradation under moist conditions and low soil pH. Conversely, IMI herbicide activity is more rapidly dissipated under neutral to alkaline soil conditions, probably a result of greater soil adsorption at lower pH (Stougaard et al. 1990). Herbicide degradation is also enhanced by higher precipitation accumulation after herbicide application (Fuesler and Hanafey 1990; Goetz et al. 1990). Soil pH can vary greatly between as well as within fields (Kells and Renner 1995); for this reason, many herbicide labels include a bioassay requirement in addition to rotation interval restrictions to determine whether the herbicide activity in the soil has decreased below levels toxic to the crop of interest. Biotechnology has the potential to be a value to production agriculture through the development of herbicide-resistant crops. Dilemmas such as concern about herbicide carryover injury to susceptible crops versus the desire and necessity for effective weed control in rotational crops can be avoided by decreasing the sensitivity of the crop to the herbicide(s) of concern. Sugarbeets are a particularly attractive target crop. Lengthy rotation restrictions for sugarbeets can severely restrict the crops and herbicides used in rotation. By reducing crop sensitivity to herbicide residues, flexibility in crop rotations can be increased and choices for chemical weed control in rotational mom can be 12 expanded. Additionally, by increasing crop safety to a currently-registered, proven herbicide, weed control options may be increased by including the herbicide into the crop protection plan in the resistant crop. Importance of sugarbeet. Sugarbeet and sugarcane are the two important crop sources of refined sucrose. Sucrose (or table sugar) serves to sweeten and preserve foods and beverages. Sugarbeet is a relatively new crop which gained acceptance and wide exposure in Europe during the Napoleonic wars in the early nineteenth century. During the wars, continental Europe was isolated from its sugar source, sugarcane, which was grown entirely in the tropical and subtropical colonies (Cooke and Scott, 1993). This necessitated the development of an alternative sucrose source that could be grown in the cooler climes of the continent. Sugarbeets are grown in temperate climates, with the majority grown in Europe, North America, and Asia. Approximately 37% of refined sugar worldwide currently is produced from sugarbeet while the remaining 63% is derived from sugarcane (Cooke and Scott 1993). The United States produces more than 25 million tons (approximately 7%) of the global sugarbeet crop on 1.4 million acres (Cooke and Scott 1993; Anonymous 1996) yielding approximately 4 million tons of refined sugar annually. Over one-half of the sugar produced in the United States comes from sugarbeets (Anonymous 1996). Michigan is the fourth largest sugarbeet producing state in the US. with approximately 187,000 acres harvested (Anonymous 1996). The value of Michigan sugarbeet production exceeds 100 million dollars annually (Fedewa and Pscodna 1994). Sugar processing and other value- added effects of sugarbeet production further benefit the Michigan economy. 13 Sugarbeet is a labor and management intensive crop to produce. The plants are low-growing and compete poorly with taller, faster growing weeds. Sugarbeet traditionally required a great deal of hand labor to thin plant populations, remove weeds, and harvest roots. Much of the hand labor has been replaced by several technological advances in the past 40 years. Sugarbeet stand thinning has become less necessary with the use of monogerm seed introduced in 1957 (Winner 1993). Mechanization of planting and harvesting has increased sugarbeet production efficiency. Average labor requirements have been reduced 12-fold from 300 man h ha'1 in 1954 to as little as 25 man h ha" today (Cooke and Scott 1993). Sugarbeet is still an intensively managed crop due to its susceptibility to many diseases and its noncompetitive nature (Dexter 1995). Sugarbeet seedlings are weak and susceptible to stand loss due to unfavorable soil moisture conditions and soil crusting, wind damage, and insect or disease damage (Schweizer and Dexter 1987). Competition from weeds is another major source of yield loss and weed control costs are extremely high in sugarbeets. In severe cases, competition from weeds can result in complete crop failure if not controlled (Schweizer and Dexter 1987). Sugarbeet weed control. Advances in establishing new plant stands and combined use of mechanical cultivation and herbicides have increased sugarbeet yields and reduced the labor cost involved with handweeding fields. Current weed control strategies for sugarbeets include a combination of PRE or PPI herbicide treatments with split applications of banded POST herbicides plus between the row cultivation (Schweizer and May 1993; Dexter 1994; Griffiths 1994). POST herbicides applied at reduced rates in split applications 5 to 7 days apart provides equivalent or greater weed control than provided by a single full-rate application of phenmedipham plus desmedipham (Dexter 1994). 14 Additionally, sugarbeets have a low margin of safety to most herbicides and the split application allows sugarbeets to effectively detoxify the lower herbicide doses without injury. Presumably the 5- to 7-day interval is long enough to allow sugarbeets to detoxify the lower herbicide close but short enough that weeds cannot metabolize the herbicide and are actually sensitized to the second dose by the first application (Dexter 1994). Herbicide damage to sugarbeet can occur when the detoxification mechanism in sugarbeets is overloaded by higher doses or herbicide activity is enhanced by spray adjuvants (Dexter 1994; Starke et al. 1996). Temperature, soil moisture, and humidity can also affect the potential injury to sugarbeets following herbicide application (Schweizer and Dexter 1987). Severe sugarbeet injury with phenmedipham + desmedipham (and other herbicides) can occur if applied on hot days with high humidity (Schweizer and Dexter 1987). Herbicide manufacturer screening programs have not been particularly successful in developing herbicidal compounds with broad spectrum weed control and an acceptable margin of safety for sugarbeet. Herbicide development programs have traditionally conducted random testing of compounds looking for chemicals that selectively kill target weeds without harming the desired crops. The difficulty lies in the effort and expense of synthesizing and testing tens of thousands of compounds in order to find one which affects target weeds but not the desired crop. Additionally, great expense is incurred to patent, develop and test experimental compounds for weed control efficacy, crop safety, and mammalian toxicological properties. The cost of discovering and bringing a new herbicide to market can be fi'om 20 to 40 million dollars (McWhorter 1987). Alternatively, weed control programs could be designed to advantageously utilize current herbicide chemistry that has 1 5 proven eficacious on weeds of a given crop, even if crop safety is in question. The use of any given herbicide is limited by selectivity between weeds and the crop. Biotechnology has the potential to increase crop selectivity by reducing the herbicide sensitivity in the crop. Additionally, focus on environmentally and toxicologically favorable chemicals may be targeted for future use in herbicide resistant crops (Mazur and F alco 1989). The development of herbicide resistant sugarbeets would be an excellent application of biotechnology to agricultural production. Weed control options in sugarbeets could be increased if sugarbeets were developed to be resistant to herbicides known to control problem weeds particular to sugarbeets like Amaranthus spp., common lambsquarters (Chenopadium album L.), Palyganum spp., and kochia [Kachia scaparia (L.) Schrad.] (Schweizer and Dexter 1987). Additionally, the risk of carryover injury could be reduced if sugarbeets were created that were resistant to the widely used, highly active, residual herbicides like the ALS-inhibiting herbicides. Increasing crop safety to herbicide carryover residues is a worthwhile efl‘ort since sugarbeet is a high value crop usually representing the highest profit potential in a given crop rotation (Schweizer and May 1993). Resistance to ALS-inhibiting herbicides may shorten or eliminate the rotation restrictions for sugarbeets (Table 2) and may increase flexibility in three or four year crop rotations currently used by sugarbeet producers (Schweizer and Dexter 1987; Dexter 1994). Crops resistant to ALS-inhibiting herbicides. By the combined efforts of plant biotechnology and traditional breeding, the designed resistance of crop plants to target herbicides has become a reality. Many herbicide resistant crops have been developed to a variety of different herbicide classes and modes of action. As the science of plant 16 transformation and tissue culture techniques were improved, attempts to decrease plant sensitivity to specific herbicides progressed beyond the model systems of tobacco and Arabidopsis (Chalefi‘ 1980; Chalefi' and Ray 1984; Haughn and Sommerville 1986, 1990; Haughn et al. 1988; Gabard et al. 1989; Harms et al. 1992). Herbicide resistance has been conferred to corn (Anderson and Georgeson 1989; Newhouse et al. 1991; Bright et al. 1992), rice (Li et al. 1992; Croughan 1996), wheat (Newhouse et al. 1992), soybeans (Sebastian et al. 1989), tomato (Lycapersican escuIentum Mill.) (Iler et al. 1993), sugarbeet (Saunders et al. 1992; D’Halluin et al. 1992), and many others. Resistances to herbicides with wide ranging modes of action have been achieved including: amino acid analogues (Harms et al. 1982; Shah et al. 1986; Hauptmann et al. 1988; Dyer et al. 1988), ACCase-inhibiting herbicides (Parker et al. 19903, 1990b), photosystem II-inhibiting herbicides (Beversdorf et a1. 1988; Stalker et al. 1988), auxin analogue herbicides (Oswald et al. 1977; Chaleff 1980) and ALS-inhibiting herbicides (McHughen 1989; Sebastian et al. 1989; D’Halluin et al. 1992; Saunders et al. 1992; Newhouse et al. 1992). The lists above do not exhaustively describe development of herbicide resistance crops but are included to indicate that designed resistance is possible with most major (and minor) crops to a wide range of herbicides. As previously mentioned, sugarbeets have a major economic impact in Michigan, as well as other sugarbeet productionregions in the US. like the Red River Valley of Minnesota and North Dakota, irrigated plains of Colorado, Wyoming, Nebraska, Idaho, and Montana, and the Pacific states of California, Washington, and Oregon (Cooke and Scott 1993; Anonymous 1996). Difficulties with weed control in sugarbeets, the need for effective broad-spectrum herbicides in sugarbeets, and the restrictive effects of herbicide {0131 site on d [hes We. the pan sun hart ALE Sl', 01m 199 Pits hart 17 rotation intervals on rotational crop weed control choices, and the threat of herbicide carryover injury to sugarbeets has stimulated researchers to attempt the development of sugarbeets resistant to ALS-inhibiting herbicides. The remainder of this review will focus on development of ALS-inhibitor resistance with intention to derive sugarbeets resistant to these herbicides. Weed resistance to ALS-inhibiting herbicides. A plant species that survives a dose of a chemical that is typically lethal to other plant species is considered tolerant to the particular herbicide; however, when a subpopulation of a normally sensitive plant species survives a lethal dose of a herbicidal compound, the isolated plants are considered herbicide resistant (Penner 1994). The potential for development of weed resistance to ALS-inhibiting herbicides became apparent only 5 years after the introduction of the first SU, chlorsulfirron, with the discovery of a chlorsulfirron-resistant isolate of prickly lettuce (Lactuca sem'ala L.) (Mallory-Smith et al. 1990a). Since then, many other weed accessions have been identified as resistant to ALS-inhibiting herbicides. A common feature to nearly all resistances derived from natural populations involved the reliance on only ALS-inhibiting herbicides for weed control (Primiani et al. 1990; Mallory-Smith et al. 1990a; Hall et al. 1990; Saari et al. 1992; Schnritzer et al. 1993). The increased selection pressure applied on natural populations by the residual activity of ALS-inhibiting herbicides is believed to greatly increase the speed with which significant populations of resistant populations appeared (Saari et a1. 1994). Table 3 presents 17 weed species reported to be resistant to at least one ALS-inhibiting herbicide. Of these, 13 are broadleaf weeds and four are monocots. The predominant mechanism of resistance has been a reduced sensitivity of the target ALS enzyme to inhibition by the herbicide. A 19 has been reported in several species (Devine et al. 1991; Hart et al. 1992; Saari et al. 1992). TP and PTB herbicides are relatively new chemistries and cross resistance tests to these classes with many herbicide-resistant isolates has not been completed; thus, the herbicide cross-class resistances described above have often been described as 1) SU- specific resistance (SU-R)‘; 2) IMI-specific resistance (IMI-R) 1; and 3) broad cross resistance (SU-R/IMI-R) 1. Imparting ALS-inhibiting herbicide resistance to crops. A broad spectrum of weeds are controlled in most of the major crops using one or more ALS-inhibiting herbicides (Table 1). Producers and chemical manufacturers would both benefit from expanded use of these chemicals in other crops and preservation of their current markets; however, crop selectivity often has been a problem with minor crops and sensitive rotational crops. Random herbicide screening procedures are designed to identify herbicides with selectivity in one or more major crops and potential selectivity in minor crops is of secondary importance. The design and development of herbicide resistance in various secondary crop species could impart the necessary margin of safety for use of the target herbicide in any crop of interest. Additionally, herbicide carryover injury could be avoided with crops resistant to ALS-inhibiting herbicides used for weed control in other crops grown in rotation. Many researchers have reported on the intentional development of resistance to ALS-inhibiting herbicides. Table 4 lists various plant species (including crops) with intentionally selected herbicide resistance. Plants resistant to ALS-inhibiting herbicides have been developed by somatic cell selection, mutation breeding, plant transformation, and interspecific crossing. 13 Sugarbeet is a labor and management intensive crop to produce. The plants are low-growing and compete poorly with taller, faster growing weeds. Sugarbeet traditionally required a great deal of hand labor to thin plant populations, remove weeds, and harvest roots. Much of the hand labor has been replaced by several technological advances in the past 40 years. Sugarbeet stand thinning has become less necessary with the use of monogerm seed introduced in 1957 (Winner 1993). Mechanization of planting and harvesting has increased sugarbeet production efficiency. Average labor requirements have been reduced 12-fold fiom 300 man h ha'l in 1954 to as little as 25 man h ha'l today (Cooke and Scott 1993). Sugarbeet is still an intensively managed crop due to its susceptibility to many diseases and its noncompetitive nature (Dexter 1995). Sugarbeet seedlings are weak and susceptible to stand loss due to unfavorable soil moisture conditions and soil crusting, wind damage, and insect or disease damage (Schweizer and Dexter 1987). Competition from weeds is another major source of yield loss and weed control costs are extremely high in sugarbeets. In severe cases, competition from weeds can result in complete crop failure if not controlled (Schweizer and Dexter 1987). Sugarbeet weed control. Advances in establishing new plant stands and combined use of mechanical cultivation and herbicides have increased sugarbeet yields and reduced the labor cost involved with handweeding fields. Current weed control strategies for sugarbeets include a combination of PRE or PPI herbicide treatments with split applications of banded POST herbicides plus between the row cultivation (Schweizer and May 1993; Dexter 1994; Griffiths 1994). POST herbicides applied at reduced rates in split applications 5 to 7 days apart provides equivalent or greater weed control than provided by a single full-rate application of phenmedipham plus desmedipham (Dexter 1994). 14 Additionally, sugarbeets have a low margin of safety to most herbicides and the split application allows sugarbeets to effectively detoxify the lower herbicide doses without injury. Presumably the 5- to 7-day interval is long enough to allow sugarbeets to detoxify the lower herbicide dose but short enough that weeds cannot metabolize the herbicide and are actually sensitized to the second dose by the first application (Dexter 1994). Herbicide damage to sugarbeet can occur when the detoxification mechanism in sugarbeets is overloaded by higher doses or herbicide activity is enhanced by spray adjuvants (Dexter 1994; Starke et a1. 1996). Temperature, soil moisture, and humidity can also affect the potential injury to sugarbeets following herbicide application (Schweizer and Dexter 1987). Severe sugarbeet injury with phenmedipham + desmedipham (and other herbicides) can occur if applied on hot days with high humidity (Schweizer and Dexter 1987). Herbicide manufacturer screening programs have not been particularly successfiil in developing herbicidal compounds with broad spectrum weed control and an acceptable margin of safety for sugarbeet. Herbicide development programs have traditionally conducted random testing of compounds looking for chemicals that selectively kill target weeds without banning the desired crops. The difficulty lies in the effort and expense of synthesizing and testing tens of thousands of compounds in order to find one which affects target weeds but not the desired crop. Additionally, great expense is incurred to patent, develop and test experimental compounds for weed control efficacy, crop safety, and mammalian toxicological properties. The cost of discovering and bringing a new herbicide to market can be from 20 to 40 million dollars (McWhorter 1987). Alternatively, weed control programs could be designed to advantageously utilize current herbicide chemistry that has 15 proven eficacious on weeds of a given crop, even if crop safety is in question. The use of any given herbicide is limited by selectivity between weeds and the crop. Biotechnology has the potential to increase crop selectivity by reducing the herbicide sensitivity in the crop. Additionally, focus an environmentally and toxicologically favorable chemicals may be targeted for future use in herbicide resistant crops (Mazur and Falco 1989). The development of herbicide resistant sugarbeets would be an excellent application of biotechnology to agricultural production. Weed control options in sugarbeets could be increased if sugarbeets were developed to be resistant to herbicides known to control problem weeds particular to sugarbeets like Amaranthus spp., common lambsquarters (Chenapodium album L.), Palyganum spp., and kochia [Kochia scaparia (L.) Schrad.] (Schweizer and Dexter 1987). Additionally, the risk of carryover injury could be reduced if sugarbeets were created that were resistant to the widely used, highly active, residual herbicides like the ALS-inhibiting herbicides. Increasing crop safety to herbicide carryover residues is a worthwhile effort since sugarbeet is a high value crop usually representing the highest profit potential in a given crop rotation (Schweizer and May 1993). Resistance to ALS-inhibiting herbicides may shorten or eliminate the rotation restrictions for sugarbeets (Table 2) and may increase flexibility in three or four year crop rotations currently used by sugarbeet producers (Schweizer and Dexter 1987; Dexter 1994). Crops resistant to ALS-inhibiting herbicides. By the combined efforts of plant biotechnology and traditional breeding, the designed resistance of crop plants to target herbicides has become a reality. Many herbicide resistant crops have been developed to a variety of different herbicide classes and modes of action. As the science of plant 16 transformation and tissue culture techniques were improved, attempts to decrease plant sensitivity to specific herbicides progressed beyond the model systems of tobacco and Arabidopsis (Chalefi‘ 1980; Chalefi‘ and Ray 1984; Haughn and Sommerville 1986, 1990; Haughn et al. 1988; Gabard et al. 1989; Harms et al. 1992). Herbicide resistance has been conferred to corn (Anderson and Georgeson 1989; Newhouse et al. 1991; Bright et al. 1992), rice (Li et al. 1992; Croughan 1996), wheat (Newhouse et al. 1992), soybeans (Sebastian et al. 1989), tomato (Lycapersican esculentum Mill.) (Iler et a1. 1993), sugarbeet (Saunders et al. 1992; D’Halluin et al. 1992), and many others. Resistances to herbicides with wide ranging modes of action have been achieved including: amino acid analogues (Harms et al. 1982; Shah et al. 1986; Hauptmann et al. 1988; Dyer et al. 1988), ACCase-inhibiting herbicides (Parker et al. 1990a, 1990b), photosystem II-inhibiting herbicides (Beversdorf et al. 1988; Stalker et al. 1988), auxin analogue herbicides (Oswald et al. 1977; Chaleff 1980) and ALS-inhibiting herbicides (McHughen 1989; Sebastian et al. 1989; D’Halluin et al. 1992; Saunders et al. 1992; Newhouse et al. 1992). The lists above do not exhaustively describe development of herbicide resistance crops but are included to indicate that designed resistance is possible with most major (and minor) crops to a wide range of herbicides. As previously mentioned, sugarbeets have a major economic impact in Michigan, as well as other sugarbeet production-regions in the US. like the Red River Valley of Minnesota and North Dakota, irrigated plains of Colorado, Wyoming, Nebraska, Idaho, and Montana, and the Pacific states of California, Washington, and Oregon (Cooke and Scott 1993; Anonymous 1996). Difficulties with weed control in sugarbeets, the need for effective broad-spectrum herbicides in sugarbeets, and the restrictive effects of herbicide 17 rotation intervals on rotational crop weed control choices, and the threat of herbicide carryover injury to sugarbeets has stimulated researchers to attempt the development of sugarbeets resistant to ALS-inhibiting herbicides. The remainder of this review will focus on development of ALS-inhibitor resistance with intention to derive sugarbeets resistant to these herbicides. Weed resistance to ALS-inhibiting herbicides. A plant species that survives a dose of a chemical that is typically lethal to other plant species is considered tolerant to the particular herbicide; however, when a subpopulation of a normally sensitive plant species survives a lethal dose of a herbicidal compound, the isolated plants are considered herbicide resistant (Penner 1994). The potential for development of weed resistance to ALS-inhibiting herbicides became apparent only 5 years after the introduction of the first SU, chlorsulfirron, with the discovery of a chlorsulfuron-resistant isolate of prickly lettuce (Lactuca serriala L.) (Mallory-Smith et al. 1990a). Since then, many other weed accessions have been identified as resistant to ALS-inhibiting herbicides. A common feature to nearly all resistances derived from natural populations involved the reliance on only ALS-inhibiting herbicides for weed control (Prinriani et al. 1990; Mallory-Smith et al. 1990a; Hall et al. 1990; Saari et al. 1992; Schmitzer et al. 1993). The increased selection pressure applied on natural populations by the residual activity of ALS-inhibiting herbicides is believed to greatly increase the speed with which significant populations of resistant populations appeared (Saari et al. 1994). Table 3 presents 17 weed species reported to be resistant to at least one ALS-inhibiting herbicide. Of these, 13 are broadleaf weeds and four are monocots. The predominant mechanism of resistance has been a reduced sensitivity of the target ALS enzyme to inhibition by the herbicide. A 18 second mechanism of resistance is increased herbicide metabolism resulting in a rapid detoxification of the herbicide and protection fi'om herbicide action. This mechanism generally has resulted in a low magnitude of cross resistance to herbicides with very difi'erent modes of action (Hall et al. 1994). Rigid ryegrass (Lalium rigidum L.) and slender foxtail (a.k.a. blackgrass) (Alapecurus myasuraides Huds.) have gained resistance to multiple modes of action‘due to enhanced herbicide metabolism (Kemp et al. 1990; Moss and Cussans 1991; Christopher at al. 1991; Holtum et al. 1991; Cotterrnan et al. 1992). These resistant biotypes interestingly were not selected using ALS-inhibiting herbicides, but enhanced metabolism was selected by repeated use of ACCase-inhibitors in rigid ryegrass (Christopher at al. 1991) and photosystem H inhibitors in blackgrass (Kemp et al. 1990). Altered target site resistance, alternatively, often results in a high magnitude of resistance specific to herbicides with a similar mode of action. In general, resistance due to an altered ALS can be classified into three types based on herbicide class cross resistance: 1) SU- and TP-resistant; 2) [ML and PTB-resistant; and 3) SU-, IMI-, TP-, and PTB-resistant (broad cross resistant). In several cases, a low level of IMI-resistance has coincided with the SU— and TP-resistant type; but, the resistance has typically been less than 10-fold and not consistent among various IMI herbicides (Saari et al. 1994). Tables 3 and 4 present cross-class resistances possessed by various weeds selected fiom wild populations and laboratory-derived resistant plants resistant to ALS-inhibitors, respectively. Resistance to one compound of a particular class of ALS-inhibiting herbicides has not guaranteed cross resistance to all members of that chemistry family. This is particularly true of the SU herbicides for which differential resistance (or lack of) 19 has been reported in several species (Devine et al. 1991; Hart et a1. 1992; Saari et al. 1992). TP and PTB herbicides are relatively new chemistries and cross resistance tests to these classes with many herbicide-resistant isolates has not been completed; thus, the herbicide cross-class resistances described above have often been described as 1) SU- specific resistance (SU-R)‘; 2) IMI-specific resistance (IMI-R) 1; and 3) broad cross resistance (SU-R/lMI-R) ‘. Imparting ALS-inhibiting herbicide resistance to crops. A broad spectrum of weeds are controlled in most of the major crops using one or more ALS-inhibiting herbicides (Table 1). Producers and chemical manufacturers would both benefit from expanded use of these chemicals in other crops and preservation of their current markets; however, crop selectivity often has been a problem with minor crops and sensitive rotational crops. Random herbicide screening procedures are designed to identify herbicides with selectivity in one or more major crops and potential selectivity in minor crops is of secondary importance. The design and development of herbicide resistance in various secondary crop species could impart the necessary margin of safety for use of the target herbicide in any crop of interest. Additionally, herbicide carryover injury could be avoided with crops resistant to ALS-inhibiting herbicides used for weed control in other crops grown in rotation. Many researchers have reported on the intentional development of resistance to ALS-inhibiting herbicides. Table 4 lists various plant species (including crops) with intentionally selected herbicide resistance. Plants resistant to ALS-inhibiting herbicides have been developed by somatic cell selection, mutation breeding, plant transformation, and interspecific crossing. 20 Somatic cell selection is a technique that attempts to isolate cells with a rare trait such as herbicide resistance from a very high number of individual plant cells or cell clumps originating fi'om the same source material. Somatic cell selection is used to select for inherent genetic variation at the cellular level which cannot easily be done at the whole plant level (Marshall 1991; Larkin and Scowcrofi 1981; Hart et al. 1994). To do so, a portion of the desired plant is induced to grow in an undifferentiated form by manipulation of the plant hormone or hormone analogue complement in a growth medium. In the case of herbicide resistance, cells are grown in suspension cultures with herbicide added or plated as a thin cell layer onto solid growth media containing the selection agent (herbicide). In time, herbicide resistance is usually displayed as the growth of one callus colony on a plate of dead cells if the selection agent concentration was acutely toxic (Anderson and Georgeson 1989; Saunders et al. 1992); or as rapid cell grth among plant cells in stasis when a chronic dose of herbicide is applied for selection (Harms et al. 1992; Iler et al. 1993). Through a series of established tissue culture manipulations, plants can be regenerated from derivatives of a single cell possessing herbicide resistance provided totipotency was present in the starting gerrnplasm and has not been lost during the culturing process. Plants can then be crossed to transfer resistance to new material by traditional breeding techniques or self-pollinated to allow identification of individuals homozygous for the resistance trait (Chalefi‘ and Ray 1984; Saunders et al. 1992). In theory, this process allows for the selection of a single cell among many thousands of purportedly identical cells which have suffered a spontaneous mutation providing an adaptive phenOtype (herbicide resistance). Current theory suggests that spontaneous mutations are the result of the cell culturing process (Larkin and Scowcroft 1981). 21 Mutation fiequency can be increased by the addition of a chemical mutatgen to the treatment and selection process. Swanson et al. (1989) mutagenized microspore cultures in a successful attempt to develop SU-R canola Two mechanisms of ALS-inhibitor resistance have been achieved by somaclonal selection. First is the most common, the selection of cells expressing an ALS enzyme with reduced herbicide sensitivity. For example, several IMI-resistant (IMI-R) corn selections were derived by acute selection on imazquin, imazethapyr, or imazapyr (Anderson and Georgeson 1989). Corn possesses two different copies of the ALS gene and among five successfully regenerated plants, herbicide resistance alleles for each locus were recovered (Anderson and Georgeson 1989; Newhouse et al. 1991; Fang et al. 1992). Corn plants specifically resistant to IMI herbicides (e.g., XI-12 and QJ-22, Table 4) or cross resistant to SU, IMI, TP, and PTB herbicides (e.g., XA-17, Table 4) were recovered (Anderson and Georgeson 1989; Bemasconi et al. 1995). A second mechanism of resistance has resulted from the increased production of the wildtype, sensitive ALS enzyme. Resistance can thus be achieved by introducing enough enzyme that although part of the enzyme population is bound and inhibited by applied herbicides, some enzyme is fiee and allowed to catalyze the normal reaction. Montoya et al. (1990) describe methods by which ALS-inhibitor resistance can be achieved by overproduction of a wildtype ALS enzyme. They claim overproduction can be due to increased gene expression caused by alterations in the gene regulatory sequence or a result of gene amplification. As shown by Harms et al. (1992), cells can be selected that simultaneously display both mechanisms of resistance. The tobacco cells were first selected for resistance to an acutely toxic concentration of cinosulfirron and subsequently 22 selected on increasing concentrations of primisulfuron in a step-wise fashion. Tobacco cells were also cross resistant to the IMI herbicide irnazaquin. ALS specific activity was six to seven times greater in the resistant cells than in wildtype cells. IMI resistance with one set of callus was lost after callus had been cultured for an extended period on herbicide-free medium. SU-R in these cells was still present. Southern blot analysis of cultures maintained with and without a selective agent indicated the IMI-R cultures that had been maintained on primisulfuron-modified media had approximately 20-fold amplification of the SurB ALS gene. Gene amplification was unstable, however, and was reduced to about a three- to four-fold level of amplification in cultures maintained in herbicide-free medium. Low magnitude of resistance in these cultures was presumably due to a mutation in the SurB ALS gene equivalent to mutations yielding SU-specific resistance in other species (Haughn et al. 1988; Guttieri et al. 1992). Other subcultures, however, had stably maintained a 20-fold gene amplification and a higher level of herbicide resistance. Variability in stability of the gene amplification phenomenon can likely be explained by an analogous drug resistance mechanism in animal cell cultures where resistance is due to the production of extrachromosomal, nuclear-localized double minute chromosomes containing only portions of a single chromosome (Stark and Wahl 1984). Maintenance of cells containing the amplified target gene on selection provided a selective advantage for cells to maintain the double minute chromosomes. If the selection agent was removed, extrachromosomal gene amplification was lost as well as the selective advantage when cultures are placed onto selective media again. Stable gene amplification can also be achieved by incorporation of double minute chromosomes into chromosomes 23 (Stark and Wahl 1984). At this point, resistance is considered genetically stable. Caretto et al. (1994) reported stable ALS gene amplification encoding a wildtype, sensitive form of the Daucus carota L. enzyme. Gene amplification resulted in chlorsulfirron resistant plants. Mutation breeding has also been used for developing ALS-resistant plants. This process attempts to select for a novel trait at the whole plant level, utilizing genetic variation induced by chemical or physical mutagenesis. Soybean seeds were treated with chemical mutagens like ethyl methane sulfonante (EMS) l and N-nitroso-N-methylurea (NMU) 1 and SU-R was selected in the M3 generation by soil-drench treatments of chlorsulfirron (Sebastian et al. 1989; Sebastian 1992). IMI-R corn was develop by pollen mutagenesis with EMS and subsequent pollination of a de-tasseled female plant (Bright et al. 1992). M1 plants were selected for IMI-R by PRE soil treatment with imazethapyr. In both cases, resistance was due to insensitive ALS enzymes. Altered ALS traits have been inherited as dominant or semidominant traits in nearly all cases reported to date (Chaleff and Ray 1984; Haughn et al. 1986; Sebastian et al. 1989; Newhouse et al. 1991, 1992; Hart et al. 1993). Interspecific crossing as a method for developing herbicide resistant crops was described by Mallory-Smith et al. (1990b) and Thill (1993). The discovery of chlorsulfuron-resistant prickly lettuce provided a genetic source of SU-R (Mallory-Smith et al. 1990a). Interspecific hybridization allowed the transfer of the resistance gene to cultivated ‘Bibb’ lettuce (Lactuca sativa L.). The resistance trait was stable in the new germplasm and was also inherited as a semidominant trait (Mallory-Smith et al. 1990b). 24 Finally, ALS-resistant plants have been developed by plant transformation with isolated ALS genes coding for ALS with decreased herbicide sensitivity. Often the source of the resistance gene has been derived previously by somaclonal selection and subsequently cloned (Chaleff and Ray 1984; Haughn et al. 1986, 1990; Creason and Chalefi‘ 1988) or by site mutagenesis of wildtype genes to achieve the same mutations as observed in the above-listed ALS genes (Wiersrna et al. 1989). Novel changes in the ALS enzyme were design based on a molecular model of the ALS enzyme and achieved by site directed mutagenesis of a wildtype Arabidapsis ALS gene with subsequent transformation (Ott et al. 1996; Kakefuda et al. 1996). A more detailed description of this molecular model will follow. ALS mutations associated with herbicide resistance. Understanding the molecular basis for plant resistance to ALS-inhibiting herbicides can be very important for several reasons. First, three different types of cross-class resistances have been observed which can have implications regarding the ability to control resistant weeds with ALS-inhibitors of difi‘erent classes (Table 3), the safety of ALS-resistant crops to different classes of ALS-inhibitor herbicides (Table 4), and the patentability of a particular resistance trait (Dietrich et al. 1992; Bright et al. 1992; Bedbrook et al. 1995; Kakefuda et al. 1996). Much of the initial experimentation with ALS was conducted in microbial or lower eukaryotic systems and was concerned with the basic biochemistry of the branched-chain amino acid pathway. Initial experiments with ALS-inhibitors also used these systems to study the effects of these herbicides on the ALS enzyme and to discover ALS-mutations resulting in herbicide resistance; however, more recent research has focused primarily on higher plant systems. 25 Yeast (Sacha-omyces cerevr'sr‘ae) served as a model system for DuPont researchers to select and molecularly characterize SU-R eukaryotic mutants (F alco and Dumas 1985; Yadev et al. 1986; Bedbrook et al. 1995). In vitra selection with chlorsulfuron or sulfometuron yielded yeast isolates resistant to SU herbicide(s). The ALS gene was isolated and sequenced from many separate isolates and the sequences compared to the wildtype, SU-sensitive yeast sequence. This process identified 10 amino acid residues within the yeast ALS enzyme associated with SU-R (Yadev et al. 1986; Mazur and Falco 1989; Bedbrook et a1. 1995). When compared to ALS sequences from other species including bacterial large subunits (Squires et a1. 1983; Wek et al. 1985; Lawther et al. 1987), and several plant species (Mazur et al. 1987; Wiersma et al. 1989; Rutledge et al. 1991; Fang et al. 1992; Grula et al. 1995; Bemasconi et al. 1995; Woodworth et al. 1996a) these ten amino acids represented highly conserved anrino acids with three regions of high homology (Mazur and Falco 1989; Hartnett et al. 1991; Bedbrook et al. 1995). A more comprehensive alignment is shown in Figure 3 grouped by higher plants, yeast, and bacterial sequences was developed using the PILEUP function of the GCG sequence analysis package. Functional equivalent amino acids (FEAA)l (strictly conserved amino acids among yeast and plant sequences) that have been associated with herbicide resistance are indicated on the first line of the sequence alignments. Since the initial work with yeast, four additional conserved amino acids (representing a total of 14 unique amino acid residues) have since been discovered to be associated with herbicide resistance in plants and are also included in the FEAA line above the aligned sequences in Figure 3. 26 Figure 4 graphically illustrates the 14 wildtype ALS sequence amino acids associated with herbicide resistance when the specific site is changed fi'om the wildtype sequence. The amino acid residues are indicated in their relative positions within the primary ALS protein structure. The shaded region, indicated by OTP‘, represents organellar transit peptide sequences responsible for direction of the nuclear-coded pro- ALS protein to the appropriate organelle, mitochondria for yeast and chloroplasts for plants, where the transit peptide is cleaved to form the mature ALS enzyme (Mazur et al. 1987, 1989; Hartnett 1991). The OTP represent regions of fairly low homology, even among plant ALS sequences. Bacteria do not have similar transit peptides. To date, herbicide resistance selection has been associated with changes at 10 different amino acids in yeast, five in plants (including one site different than in yeast), and two sites in bacteria (Figure 4). Three highly conserved amino acid residues unique from the 11 residues described above were chosen for site directed mutagenesis and shown to cause ALS-based herbicide resistance when transformed into plants. These sites were chosen as potentially important amino acids in the enzyme active site based on a molecular model of the ALS protein (Kakefirda et al. 1996; Ott et al. 1996). These sites are indicated in Figure 4 as “rationally-designed sites.” Many selections of ALS-inhibiting herbicide resistance in the field and in the laboratory have already been described. Herbicide resistance could be classified as SU- specific, IMI-specific, and broad cross-class resistance. The ALS genes from many different herbicide-resistant selections have been sequenced and shown to contain one or more changes in the ALS protein structure believed to be the cause of resistance. The substituted amino acid for each isolated herbicide resistance case is shown in Table 5 27 under the FEAA wildtype residue present for the fourteen sites indicated in Figure 4 and are listed with the corresponding cross-class resistance characterization. The most complete cross-class resistance characterization has been conducted with plant species. The corresponding amino acid sequence number for each of the herbicide resistance amino acid substitutions (Table 5) have been omitted in lieu of the FEAA number shown in Figure 4. The literature abounds in confirsion regarding the identification of analogous mutations in different species because of the variation in ALS enzyme length fi'om one organism to another. Additionally, some research articles have attempted to describe mature plant ALS enzymes by numbering fiom the putative chloroplast transit peptide cleavage site (Bemasconi et al. 1995;1(akefirda et al. 1996). To date, N-sequencing of a mature ALS fiom plants has not been reported. Uncertainty regarding the cleavage site is therefore a cause for concern. Examples of the variation of FEAA numbering systems are shown in Table 6. In this table, the 14 FEAA identified in Figures 3 and 4 and Table 5 are listed in the first column. The FEAA resistance residue number (column 2) corresponds to Figure 4 displaying these sites in order (amino to carboyxl) within the primary protein structure. The third column indicates the corresponding residue number found in the Arabidapsis pro-ALS enzyme. Arabidapsis was chosen as the model because it is the longest plant ALS sequence reported to date, making sequence alignment easier. It is also the model organism for plant genetics studies. The source organism for the characterized ALS is listed in the fourth column and the corresponding amino acid residue number with respect to the specific organism is displayed in column five. In several cases, discrepancies over amino acid number have occurred within species, even within the same article (note especially FEAA residue A2). 28 We propose to simplify the numbering system as shown in Figure 4 and in column 2 of Table 6. By aligning the sequence of a newly sequenced ALS with the Arabidapsis ALS sequence (Figure 3), one can easily identify analogous (functionally equivalent) amino acids within the new sequence. Changes from the wildtype sequence can be noted according to the FEAA numbering system which is conducive to all species rather than (or in addition to) a species specific number of the amino acid residue. This paper would serve as the benchmark for the numbering of FEAA residues associated with ALS- inhibiting herbicide resistance. The 14 FEAA residues identified to date would be numbered in consecutive order of their occurrence in the primary ALS protein structure. We also propose that, upon discovery of additional ALS mutations sites, the FEAA be numbered consecutively hereafter regardless of position in the ALS sequence. Precedence for a numbering system according to chronological order of discovery exists with the nomenclature of gibberellins (Graebe 1987) as well as glutathione-S-transferase isozymes in maize (Irzyk and Fuerst 1993). Alternatively, the Arabidopsis pro-ALS enzyme could be held up as the benchmark numbering system and firture ALS sequence changes numbered reported in terms of the species residue number as well as the Arabidapsis FEAA residue number. Either system would clearly be superior to the current state of confirsion regarding ALS-mutation sites responsible for herbicide resistance. Of the 14 sites involved in ALS-inhibitor resistance (Figure 4), 10 SU-R sites have been reported for yeast. Five of the yeast sites (FEAA residues G, K, Mg, D10, and V”) have only been described from the yeast selection system (Bedbrook et al. 1995) the other five have been selected from wildtype populations of other species as well. All 10 sites reported for yeast (6;, A2, P4, A5, Kc, M9, D10, Vu, W12, and F13) result in SU-R in yeast 29 systems (Mazur and Falco 1989; Hartnett et al. 1991; Bedbrook et a1. 1995). No data regarding cross resistance to other ALS-inhibiting herbicide classes was reported for any of these sites with the yeast system. It is clear fi'om the work of Bedbrook et al. (1995) that ALS-based SU-R in yeast requires at the minimum a change of the amino acid residue at any one of the ten sites from the normal, wildtype amino acid in order to achieve SU-R; however, difi‘erent amino acids are more or less effective in imparting SU-R on the ALS enzyme. The number of possible substitutions for any of the above 10 sites that still provides SU-R ranges from four to 18 (Figure 5). Two SU-R bacterial isolates were also identified with altered ALS proteins. The single amino acid changes were observed at the FEAA residues P4 and A; (Figure 5). IMI cross resistance was not determined for either of these mutants. Only one amino acid substitution was observed for each of these sites in bacterial systems. Plants resistant to ALS-inhibiting herbicides derived by laboratory or field selection have shown amino acid substitutions at five sites identified in Figure 4 as A2, P4, A6, W12, and Sir. IMI-specific resistance was observed in the Mississippi (MS) cocklebur (Xanthium stmmarium L.) accession, associated with a T for A2 substitution (Bemasconi et al. 1995, 1996). Bright et al. (1992) described the selection of an IMI-specific resistant corn isolate (mutant 1) derived by pollen mutagenesis that also showed a T for A2 substitution. Bemasconi et al. (1995) described ‘mutant 1’ as the source of IMI-R in the commercialized ICI-IT corn hybrids; however, a second IMI-specific resistant isolate (mutant 2) described by Bright et al. (1992) was commercialized insteadz. Mutant 2 was 2 J. A Greaves, ICI/Garst Seeds, Inc., Slater, IA. Personal communication. 30 IMI-R due to a substitution of N for S“, the second known site for IMI-specific resistance in plants. The equivalent mutation, N for S14 , was observed in two other IMI-specific resistant corn isolates, XI-12 and QJ-22, described by Anderson and Georgeson (1989) and sequenced by Dietrich (1992). Sathsavian et al. (1991) reported the equivalent substitution was present in an IMI-specific resistance in Arabidapsis. The 814 residue represents the only randomly selected amino acid substitution site (out of 11 total) that was not described as a SU-R site in yeast. To date, the N substitution is the only observed substitution at this FEAA residue. Five difi'erent substitutions have been observed at the P4 residue in plants (Table 5). These resistances are considered SU-specific, although slight resistance to IMI herbicides has also been observed (Saari et al. 1990; Mallory-Smith et al. 1990; Guttieri et al. 1992; Harms et al. 1992). Cross resistance to all SU herbicides, however, was extremely variable in plants. The S for P4 substitution in tobacco provided about three- fold resistance to primisulfurOn while resistance to chlorsulfuron was over 30-fold (Harms et al. 1992). Substitutions at P4 have been detected in isolates of Arabidapsis, kochia, tobacco, and prickly lettuce. Very high levels of resistance to all four classes of ALS-inhibiting herbicides has been observed in plants carrying the L for W12 substitution. Selection of this mutation fi'om wild populations has occurred in a Missouri (MO-1) accession of cocklebur (Bemasconi et al. 1995), common waterhemp (Amaranthus rudis L.) (Woodworth et al. 1996), and smooth pigweed (Amaranthus hybridus L.)3. Laboratory selections with this 3 R Schmenk, Univ. of Kentucky. Personal communication. 3 1 mutation include canola (Brassica napus L.) (Hattori et al. 1995), the 201-17 corn which is marketed as Pioneer IR hybrids (Bemasconi et al. 1995), and tobacco (S4-Hra) (Lee et al. 1988). The tobacco selection actually was a double mutant derived by a two step selection process in an attempt to increase the SU—R observed in an original SU-R selection, S4 (Creason and Chalefi‘ 1988). Cross resistance to non-SU herbicides was not conducted with these plants; however, SU-R was about five-fold higher in S4-Hra versus S4. Substitution of 18 difi‘erent amino acids at W12 in yeast imparted SU-R; cross resistance to IMI or other classes was not determined for the different yeast mutants (Bedbrook et al. 1995). Leucine is the only amino acid substitution observed at this site in selected herbicide-resistant plant isolates. Broad, but low levels of resistance to all classes of ALS-inhibitors have been reported in a second Missouri (MO-2) accession of cocklebur resulting fi'om a V for A6 substitution (Woodworth et al. 1996b). The level of enzyme resistance was about 10-fold to representative chemicals from four classes of ALS-inhibiting herbicides, much lower than the >1000-fold resistance provided by the broad cross resistance W12 mutations. The MO-2 cocklebur accession represents the only reported plant with a substitution at the Art FEAA residue. The equivalent mutation at the A5 site in yeast conveyed SU-R; however, neither the magnitude of resistance or extent of cross resistance was not reported (Bedbrook et al. 1995). ' Three additional amino acid substitutions can impart herbicide resistance to plant ALS enzymes; however, none have been reported in natural selections fiom any species. Site directed mutagenesis of a cloned Arabidapsis ALS gene resulted in rationally designed mutations at three amino acid residues (M3, R5, and F7) independently (Figure 5). 32 Ott et al. (1996) recently described a molecular model of a plant ALS enzyme to identify novel mutational sites for rationally designing herbicide resistance. The model of the Arabidapsis ALS homodirner was derived by homology modeling using the X-ray crystal structure of pyruvate oxidase from Lactabacillus plantarum (Muller and Schulz 1993), primary and computer-predicted secondary ALS structure, and structure-activity data fi'om previous IMI analogue testing (Ott et al. 1996). Pyruvate oxidase (P0X)l and ALS were previously suggested to have a common ancestral origin based on their biochemical and structural similarities (Chang and Cronan 1988). TPP, FAD, and Mg’2 are required for full enzymatic activity, both enzymes utilize pyruvate as a substrate, and both enzyme reactions utilize a hydroxyethyl-thiamine perphosphate intermediate mechanism to decarboxylate pyruvate (Hawkes et al. 1989; Schloss et al. 1990). Both enzymes form higher orders of subunit organization with increasing FAD and/or subunit concentration (Singh and Schmitt 1989; Dumer and BOger 1991; Risse et al. 1992). Prokaryotic POX is a homotetramer of 65 kD subunits (Grabau and Cronan 1986) whereas prokaryotic ALS is a heterotetramer of 2 large (60-65 kD) subunits and 2 small (10-17 kD) subunits (Grimminger and Umbarger 1979; Eoyang and Silverrnan 1984; Saari et al. 1994) and plant ALS has been predicted to aggregate as a homo-dimer, tetramer, or higher order of assembly (Singh et al. 1988b; Singh and Schmitt 1989; Stidham 1991; Dumer and BOger 1991). POX and ALS do differ, however. ALS catalyzes the non-oxidative condensation of two pyruvate molecules to acetolactate with concomitant release of CO2 (or the analogous condensation of pyruvate and 2- ketobutyrate) whereas POX catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2. Most flavoenzymes are involved in oxidative reactions; however, the oxidative 33 state of FAD does not affect the catalytic ability of ALS, leading investigators to propose the firnction of FAD is structural in nature (Schloss et al. 1988; Singh and Schmitt 1989; Dumer and Edger 1991). The POX active site is a solvent-filled pocket created by dirnerization of two monomers. The substrate entry site and proposed herbicide binding site for ALS was similarly predicted (Ott et al. 1996). Nearly all of the 10 SU-R sites in yeast were predicted to cluster within the proposed herbicide binding site (Ott et a1. 1996). A short a -helix containing FEAA residues Vu, W12, and F13 as well as a loop containing Gt were predicted to interact with the TPP cofactor (Kakefirda et al. 1996). K3 was considered a critical residue to interact with the negatively charged carboxyl group of acidic herbicides, acting as a herbicide “anchor” (Kakefuda et al. 1996). A2 and P4 were predicted to form a hydrophobic pore which correctly position theK. residue for herbicide binding. Presumably the positively charge Kc residue is functionally involved with acetohydroxy acid (pyruvate and 2-keto butyrate) conveyance as well as the R5 residue. G and A2 were also predicted to hydrogen bond with imazethapyr causing the methyl substitute of the irnidazole ring to interact with V11 of the short or-helix (Kakefuda et al. 1996). The ALS molecular model seemed particularly successful in accounting for much of the known herbicide resistance data, so it was used to predict additional mutations within the herbicide-binding site which could potentially result in IMI-specific resistance. A trial- and-error iterative process was effective in producing a fully active mutant Arabidopsis ALS enzyme by site directed mutagenesis that was resistant to IMI herbicides. An I for M3 mutation, E for R5 substitution, and R for F1 change each yielded IMI-R ALS enzyme (Ott et al. 1996; Kakefirda et al. 1996). Substitutions of E for M3 and A for R5 resulted in 34 - resistant enzymes; however, specific activity was unacceptably reduced by these mutations (Ott et al. 1996). In this review of ALS mutations associated with herbicide resistance, we have proposed a new numbering system for herbicide resistance mutations intended to avoid confusion and errors in the literature that are the result of ALS enzymes of differing sizes. The system utilizes functionally equivalent amino acids determined by sequence alignment of any given ALS peptide sequence with the Arabidapsis wildtype enzyme. Figure 4 displays the relative position of the currently-known ALS herbicide resistance mutation sites and their assigned FEAA residue number. Table 5 shows the corresponding resistance phenotype of plants, yeast, and bacteria for each amino acid substitution known. Table 6 displays the FEAA with its corresponding number assigned here, the Arabidapsis equivalent amino acid residue, and examples of the plethora of different numbers assigned in the literature for each of the FEAA residues. Table 7 summarizes the cross-resistance phenotypes for substitutions at each of the FEAA residues for plants, yeast, and bacteria. Cross resistance data is insufficient in yeast to make general statements for the various substitutions; however, four cross resistance classifications can be generalized in plants. 1) IMI-specific resistance was observed for amino acid substitutions for the wildtype residues at FEAA A2, R5, F1, and S14. Only the substitutions at A2 and S14 have been observed fi'om non-engineered populations. 2) SU-specific resistance occurred with amino acid substitutions at FEAA residue Pr. 3) A low level of resistance was observed to all four classes of ALS-inhibiting herbicides with amino acid alterations at M; and Ac. Of these, only A; has been selected from a natural plant population. 4) High levels of broad cross resistance to all classes of ALS-inhibitors was seen with the L for W12 mutation. No 35 other amino acid substitutions in plants has been recorded. Cross resistance data for yeast substitutions at residue W12 were not reported. Important lessons have been learned regarding the molecular structure of the ALS enzyme even though the enzyme has not been purified and its structure examined by X-ray crystalography. A combination of herbicide physiology, biochemistry, molecular biology, and homology modeling has at least partially circumvented difficulties faced using conventional biochemical techniques to purify an unstable enzyme from plants. The utility of the ALS molecular model may be to design even more herbicide-specific resistance mutations; or, perhaps the model could be utilized for designing novel chemistries capable of inhibiting ALS enzymes of weeds that have gained broad cross resistance to all of the currently developed herbicides. Statement of the Problem and Attribution. Sugarbeets are a high value crop grown in temperate regions as an alternative sucrose source to sugarcane. Sugarbeets are commonly grown in 3- to 4-year rotations with crops such as corn, dry beans or soybeans, and cereal crops. Sugarbeets can be severely injured by some of the herbicides used in these rotational craps if residues persist in the soil at a high enough concentration to effect a phytotoxic response. Sugarbeets are .extremely sensitive to nearly all ALS-inhibiting herbicides and can be injured by low levels of these chemicals. Concern about sugarbeet injury from herbicide carryover has resulted in lengthy rotation restriction intervals for ALS-inhibiting herbicides with sugarbeets. Effective chemical weed control in sugarbeets is also difiicult due to the marginal crop safety displayed by sugarbeets to many currently marketed herbicides. 36 Saunders et al. (1992) successfirlly utilized somatic cell selection procedures to obtain a novel sulfonylurea resistance trait in sugarbeet in an attempt to reduce the effect of carryover residues to sugarbeets. Our goal with the current research was to derive imidazolinone resistance and perhaps combine cross resistance to other herbicide classes in sugarbeets utilizing the somatic cell selection techniques previously employed. The magnitude and extent of resistance to different ALS-inhibiting herbicide classes were then determined for two novel somaclonal mutants, the mechanism of resistance determined, and the molecular basis for resistance elucidated. Chapter 1 contains a comprehensive review of current literature with regard to ALS-inhibiting herbicide resistance research in weeds and crops. Extensive discussion regarding the state of knowledge of specific mutations responsible for herbicide resistance has also been included. In this review, a new numbering system for ALS enzyme mutations responsible for herbicide resistance was introduced in an attempt to moderate confusion in the literature regarding equivalent mutations in different species. A portion of chapter 1 will be revised to include data derived from later chapters of this thesis and submitted as a review article on ALS herbicide resistance mutations in an appropriate forum like the Annual Review of Plant Physiology and Molecular Biology. Later chapters will be submitted to the Weed Science journal and are included here in journal format. Chapter '1 was also written in Weed Science format for consistency. Chapter 2 describes the selection of two imidazolinone-resistant sugarbeet isolates and mode of inheritance of the resistance traits. Most of the work was completed by myself under the supervision of Dr. Donald Penner, co-author of the paper to be submitted to the Weed Science journal. Bryan Young under the direction of Dr. Penner and Wendy 37 Pline under the supervision of Dr. Penner and myself were involved in the initial screening for imidazolinone resistance resulting in the discovery of Sir-13 and 93R30B, respectively. The expertise of Dr. Joseph Saunders was instrumental in the selection and breeding of herbicide resistance traits in these sugarbeets. He also supplied the source materials, REL- 1 and 93R30, for somatic cell selection. His guidance and the use of his cell culture equipment made this efi‘ort possible. Equipment and facilities for Southern blot analyses were supplied by American Cyanamid and the assistance and guidance of Dr. Newell Bascomb and Steve Stumer in molecular techniques were extremely helpfirl. Chapter 3 contains in vitro and whole plant characterization of the magnitude of imidazolinone resistance and extent of cross resistance of the two new sugarbeet somaclonal selections. The paper will be submitted to the Weed Science journal with co- authors of myself and Dr. Donald Penner. Significant contributions were provided by Beth McNeilly, Amy Gonzalez, and Wendy Pline through propagation of shoot cultures used for in vitro resistance studies. Chapter 4 details the ALS-enzyme based mechanism of resistance and the molecular basis for the altered enzyme sensitivity in the sulfonylurea-resistant selection, Sur (Saunders et al. 1992), and the two imidazolinone-resistant selections, Sir-l3 and 93R30B. The chapter will be submitted to the Weed Science journal co-authored by myself and Dr. Penner. Sequence analyses of PCR-generated ALS gene fragments were conducted at the American Cyanamid facilities in Princeton, NJ with the assistance and BUidance of Steve Stumer and Dr. Newell Bascomb. Chapter 5 briefly discusses some of the implications of this research to agriculture and the characteristics of the sugarbeets which may determine the level of their success in 38 the market. Suggestions for additional investigations with the herbicide-resistant sugarbeets are also discussed. Apendix A includes the chemical names for all ALS-inhibiting herbicides listed in this manuscript. 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Vidal, R A. and N. G. Fleck. 1997. Three weed species with confirmed resistance to herbicides in brazil (sic). Abst. Weed Sci. Amer. 37:251. 168. 169. 170. 171. 172. 173. 174. 175. 60 Walsh, J. D., M. S. Defelice, and B. D. Sims. 1993. Soybean (Glycine max) herbicide carryover to grain and fiber crops. Weed Technol. 7 2625-632. Weinstock, 0., C. Sella, D. M. Chipman, and Z. Barak. 1992. Properties of subcloned subunits of bacterial acetohydroxyacid synthases. J. Bacteriol. 174:5560-5566. Wek, RC., C. A. Hauser, and G. W. Hatfield. 1985. The nucleotide seuqence of the ilvBN operon of Escherichia coli: sequence homologies of the acetohydroxy acid synthase isozymes. Nucl. Acids Res. 3995-4010. Wiersma, P. A, M. G. Schmiemann, J. A Condie, W. L. Crosby, and M. M. Moloney. 1989. Isolation, expression and phylogenic inheritance of an acetolactate synthase gene from Brassica napus. Mol. Gen. Genet. 2192413-420. Winner, C. 1993. History of the crop. Pages 1-36 in D. A. Cooke and R. K. Scott eds. The Sugar Beet Crop. Chapman and Hall, London, England. Woodworth, A. R, B. A. Rosen, and P. Bemasconi. 1996a. Broad range resistance to herbicides targeting acetolactate synthase (ALS) in a field isolate of Amaranthus sp. is conferred by a Trp to Leu mutation in the ALS gene. Plant Physiol. 111:1353. Woodworth, A, P Bemasconi, M. Subramanian, and B. Rosen. 1996b. A second naturally occurring point mutation confers broad-based tolerance to acetolactate synthase inhibitors. Plant Physiol. 111:8105. Wright, T. R. and D. Penner. 1996. The inhibition of corn acetolactate synthase (ALS) by various ALS inhibiting herbicides. Abstr. Weed Sci. Soc. Am. 36:59. 61 176. Yadev, N., R E. McDevitt, S. Benard, and S. C. Falco. 1986. Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl. Proc. Natl. Acad. Sci., USA 83:4418-4422. 62 Table 1. Currently marketed and experimental ALS-inhibiting herbicides. Representative Primary Common name' trade name Herbicide class Manufacturer crop Amidosulfirron" Adret SU AgrEvo Cereals Azimsulfuron" - SU DuPont Rice Bensulfuron° Londax SU DuPont Rice Chlorimuron‘ Classic SU DuPont Soybeans Chlorsulfuron" Glean SU DuPont Cereals Cinosulfuron" Setoff SU Ciba Rice Cloransulam" First Rate TP DowElanco Soybeans Cyclosulfamuron" -- SU American Cyanamid Rice Ethametsulfirron" Muster SU DuPont Canola Flazasulfirron" Sibagen SU ISK Turf Flumetsulam° Broadstrike TP DowElanco Corn Halosulfillronc Permit SU Monsanto Corn Imazameth‘ Cadre IMI American Cyanamid Legumes Imazamethabenz“ Assert IMI American Cyanamid Cereals Irnazamox‘l Raptor IMI American Cyanamid Legumes Imazapyr" Aresenal IMI American Cyanamid Noncrop Imazaquin° Scepter IMI American Cyanamid Soybeans Imazethapyrc Pursuit IMI American Cyanamid Legumes Table 1. Continued. 63 Representative Primary Common name trade name Herbicide class Manufacturer crop ImazosulfuronF Takeofi’ SU Takeda Rice RIB-2023" .- POB‘ Kumiai Rice KIH-6127" .- POB Kumiai Rice Metosulam" Eclipse TP DowElanco Cereals Metsulfirron° Ally SU DuPont Cereals Nicosulfilron° Accent SU DuPont Corn Oxasulfuron" Expert SU Ciba Soybeans Primisulfirronc Beacon SU Ciba Corn Prosulfirron‘ Peak SU Ciba Corn Pyrazosulfirron" Sirius SU Nissan Rice Pyrithiobac° Staple PTB DuPont Cotton Rimsulfirronc Matrix SU DuPont C om Sulfometuron‘ Oust SU DuPont Noncrop Sulfosulfuron‘ -- SU Monsanto Wheat Thifensulfirronc Pinnacle SU DuPont Legumes 'l‘riasulfirronc Amber SU Ciba Cereals Tribenuron° Express SU DuPont Cereals Triflusulfuron‘ UpBeet SU DuPont Sugarbeets rChemical names listed in Appendix A. 64 Table 1. Continued. " Global directory of herbicides (Hopkins 1994). c Crop protection chemicals reference (Anonymous 1997). an Environmental use permit, 1996. ° Proceedings of the North Central Weed Science Society, 1996. POB‘, pyrirnidinyloxybenzoate. 65 Table 2. Rotation restrictions for sugarbeets with ALS-inhibiting herbicides (Anonymous 1997). Herbicides not listed here either do not persist to injure sugarbeets or are not used in regions where sugarbeets are grown. Requirement before Herbicide Herbicide class planting sugarbeets (months) Chlorimuron SU 30 Cloransulam TP 24 + BA‘I Flumetsulam TP 26 + BA Irnazameth IMI 40 Irnazamethabenz IMI 20 Irnazaquin .IMI 26 Imazethapyr IMI 40 + BA Metsulfirron SU 34 + BA Nicosulfirron SU 10-18b Primisulfirron SU 18 Prosulfirron SU 22 Pyrithiobac PTB 10 + BA Rimsulfuron SU 10 Thifensulfirron SU 2 Triasulfirron SU 24 + BA Tribenuron SU 2 66 Table 2. 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(1996) Zea mays 57 Bemasconi et al. (1995) Zea mays 56 Bemasconi et al. (1995) E. coli (ALS II) 26 Yadev et al. (1986) M 3 12'4 Zea mays (mature) 53 Kakefuda et al. (1996) Arabidopsis 124 Ott et al. (1996) P 4 197 S. cerevisiae 192 Yadev et al. (1986) Mazur and Falco (1989) Nicotiana tabacum 196 Lee et a1. (1988) Arabidopsis 197 Haughn et a1. (1988) Synechococcus 115 F riedberg and Seijffers (1989) Brassica napus 173 Wiersma et a1 (1989) Table 6. Continued. 83 FEAA Arabidopsi: Source FEAA residue no. residue no. ALS source residue no. Reference R 5 199 Arabidopsis 199 Ott et a1. (1996) Zea may: (mature) 128 Kakefuda et al. (1996) A 6 205 S. cerevisiae 200 Mazur and Falco (1989) X. :truman’um (MO-2) 183 Woodworth et al. (1996b) F 7 206 Arabidopsis 206 Ott et al. (1996) Zea may: (mature) 135 Kakefuda (1996) K 8 256 S. cerevisiae 251 Mazur and Falco (1989) M 9 351 S. cerevisiae 354 Mazur and Falco (1989) D 10 376 S. cerevisiae 374 Mazur and Falco (1989) V 11 571 S. cerevisiae 583 Mazur and Falco (1989) W 12 574 S. cerevisiae 586 Mazur and Falco (1989) Nicotiana tabacum 573 Lee et al. (1988) X. :truman‘um (MO-l) 552 Bemasconi et al. (1995) Zea may: 542 Bemasconi et al. (1995) Brassica napus 557 Hattori et a1. (1995) Amaranthu: :p. 569 Woodworth et al. (1996a) F 13 578 S. cerevisiae 590 Mazur and Falco (1989) S 14 653 Arabidopsis 653 Sathsavian et al. (1991) Zea may: (XI-12) 621 Dietrich (1992) Zea may: (QJ-22) 621 Dietrich (1992) Zea may: (mutZ) 621 Bright et al. (1992) 84 Table 6. Continued. FEAA Arabidopsis Source FEAA residue no. residue no. ALS source residue no. Reference Arabidopsis 656 Bemasconi et a1. (1995) 85 Table 7. Herbicide cross resistance phenotype summary amino acid substitutions from the wildtype fimctional equivalent amino acid (FEAA) for plants, yeast, and bacteria. Plant resistance Yeast resistance Bacteria resistance FEAA No. SU IM SU IM SU [M G 1 ND ND R ND ND ND A 2 S R R ND R S M 3 R R ND ND ND ND P 4 R S R ND R ND R 5 S R ND ND ND ND A 6 R R R ND ND ND F 7 S R ND ND ND ND K 8 ND ND R ND ND ND M 9 ND ND R ND ND ND D 10 ND ND R ND ND ND V 1 1 ND ND R ND ND ND W 12 R R R ND ND ND F 13 ND ND R ND ND ND 6 e 6 a 86 Figure 1. Representative compounds from four commercialized ALS-inhibiting herbicide chemical classes. c1 ocn @E—w-E-wb CH3 Sulfonylurea (chlorsulfuron) F 0 Na /\ _l| / N ‘1‘“ ESE-gag, Triazolopyrimidine (flumetsulam) 87 H O Imidazolinone (imazethapyr) O Na+ OCH3 4. Pyrimidinylthiobenzoate (pyrithiobac) Fig 88 Figure 2. Biosynthetic pathway for the branched-chain amino acids. Enzymes involved are shown in boxed italics. Site(s) of endproduct feedback regulation are indicated by dashed arrows. 89 Threonine\ Pyruvate 77veom‘ne \ + deMamse 2-Ketobutyrat Acetolactate synthase Acetohydroxybutyrate Acetolactate Acetohydroxyacid reductoisomerase Dihydroxy-methyl-valerate Dihydroxy-isovalerate Dihydroxyacid dehydratase 2-Keto-methyl-valerate 2-Keto-isovalerate l Valine aminotransferase / ' --------------- Isoleucine Valine ------------- r ------------ - " 2-180propylmalate synthase 4 ................ V E 2-Isopropylmalate 3-I:0pr0pylmalate dehydratase 3-Isopropylmalate dehycb'ogenase 3-Isopr0fylmalate 2-Keto-isocaproate Leucine aminotransferase l Leucine 90 Figure 3. Deduced amino acid sequence alignment of ALS enzymes from eight plant species, yeast, and E. coli. FEAA indicates conserved, “functional equivalent amino acid” residues associated with herbicide resistance when changed from the indicated wildtype amino acid. Amino acid numbering along the top of the aligned sequences pertains to the Arabidopsis sequence number. Dots indicate sequence gaps inserted to optimize sequence alignment. For key to one-letter amino acid abbreviations, see Appendix B. 91 mumm044¢>w Addddfifldfid A¢8m44<<8¢ mAmmMMHmHm mflmmMmHmHm BhfiflmzmhmH .....Mflm9m mmmBBMmmlm mmmBBMmmmm flammmMOmBA mummmmafifi> Admmoxomfifl nmfimfixmmfim mmmmmMZQZA mv midmazmwflH thmmBABmA mthmanfima demmOQwax BHQhBthAm mmmmfimmmgq Ahmhfimmmqq mhmflmmmmHm m>mth>mdx mhmflmmMmHm .hm......a mmmflmh&MHm monumxH=...... aeHmmzammH exam.mmmm¢ axmmemmmmm zeaanmmmm eommmmmmhou mmoxEOHucuomm mod mxua «on woa mama «on ma sausmhwn Sawmxmmow m asusuhwc sawmkmmou .mm uncucmhmew sawhufiahum ancucmk musm anmnmu mcmwuouwz «new anomnmu MGMfluoqu HHHrmzmmq newmmmhm HHumamm: MUflmmeHm Hlmzmec mowmmmhm mwheewa> euom memflqmcu mammoeflecu< ddmh 92 mBDSQQWCMA m03mmam84m QUSQMAQBQQ Qdummthmm OdeMHmDmm mmmmm>hmmm ACNMmHmmKB Athm>th¢ mdflmm>hawa mhamz m0m¢m>hmmo mdfimMHmBmm mm xxddxmmmdm m¢BQBm<< Adzxm8>m8¢ xooaflmmmmo mummmfiH... v~0,H.nm..m... MHBO..m... xnfixmm...4 zamMm....< KOBMHmmmm¢ WHdmmmm... 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Model ALS enzyme primary protein stmcture indicating relative positions of conserved amino acid residues implicated in resistance to ALS-inhibiting herbicides when changed from the wildtype amino acid corresponding to the FEAA residues in Figure 3. ALS-inhibitor resistance has been associated with changes at 14 conserved amino acid sites, 11 of which have been derived by selection from natural populations: 10 in yeast, five in plants, and two in bacteria. Three of the indicated residues were derived by site directed mutagenesis of novel resistance sites not previously selected from natural Populations. These sites were determined by ALS enzyme modeling and rational design of mutations (Ott et al. 1996; Kakefirda et al. 1996). The amino terminus represents the organellar transit peptide (OTP)l responsible for directing the pro-ALS enzyme to the appropriate organelle in eukaryotes (chloroplast in plants or mitochondria in yeast) where it is cleaved to form the mature enzyme. 106 A353 =5 8% m m 2 888-2525 < m nM.—38m m 3 . . < . m < 355 m 3 > O E M < m < O 88> 3:832 02995: 5 333.95 8:28. 28 05:2 . Emwawuo E m— 2: 2 a w e n N _ eonfiacfimm a. o m m Es» a 2 x m < m m 2 0.05. 133 Table 3. IMI-R/SU—R phenotype distribution for 93R3OB F2 herbicide resistance screen. Model tested assumes 93R3OB trait (IMI-R/SU-R) is a single nuclear dominant trait. Theoretical Observed Expected (9i): Phenotype distribution frequency frequency e Susceptible (IMI-S/SU-S) l 53 42.5 2.6 Resistant (IMI-R/SU-R) 3 117 127.5 0.9 (homozygous + heterozygous) X2 = 3.5 0.10.05. 1 34 Table 4. Chi square analysis of observed 93R3OB F2 progeny test phenotype distribution versus proposed one or two ALS loci models. Assumptions made: if one locus, 93R3OB had a IMI-R/SU-R phenotype; if two loci, 93R3OB could be either IMI-R/SU-R or IMI- R/SU-S. ---One locus-— ---- -------- --Two loci mmmmm ‘ ““"""' F1 ism-u?”- (93R3OBMI,Sur/ivt)' Observed Theoretical Expected Phenotype frequency distribution frequency IMI-R/SU-R 1 1 7 IMI-R/SU-S 0 IMI-S/SU-R O M-S/SU-S 53 0.1>P>0.05° 0.0010.05. ‘Probability of exceeding chi square when hypothesis is true. Reject proposed mode of inheritance for P<0.05. 135 Figure 1. Phenotype evaluation to determine herbicide resistance allelism. A. Expected F 1 genotype and phenotype segregation ratio, and observed F1 phenotype distribution fi'om a four allele cross. B. Possible testcross and progeny test herbicide class resistance segregation ratios for three IMI-R/SU-R F1 genotypes (Figure 1A, shaded box). C. Observed testcross and progeny test herbicide class resistance segregation ratios for three IMI-R/SU-R F1 individuals. 1B. 1C. 136 93R3OB/Sur X Sir-13A“ 1A. s x2=o.o P=1.0',df=1 Testcross (Wt/Wt X F1 Self pollinate Expected testcross Expected testcross slsss rgistance digribugign class resistance distribution IMI/SL1 IMI 511 m IMI/SQ IMI SL1 m A. 1 1 0 0 A. 3 1 0 0 B. 1 0 0 1 B. 3 0 0 1 IC. 0 1 1 0 C 2 1 1 0 Observed testcross Observed testcross slsss resistance distribugign class resistance distributign IMI/$11M s11 m x2 gr IMI/SUIMI 31.1 wt x2 df A - -- -- -- A. -- -- - - B. -- -- -- -- B. 96 0 0 22 2.5 I“ C o 27 22 o 0.5“1 C. 13 4 5 o 0.8 2“ ' Probability of exceeding chi square when hypothesis is true. Accept proposed mode of inheritance for P>0.05. " (0.5>P>0.3). ° (0.2>P>0. l). " (o.7>1>>o.5). 137 Figure 2. Sugarbeet Southern blot analysis for ALS gene copy determination. A. Southern blot. B. Restriction map for 1998 bp sugarbeet ALS gene: no internal Bam H1 or Hind III sites exist. C. Expected and observed band number and sizes for single ALS gene copy number. 138 0 Kb - 1 Kb - Eco RI «- XbaI <— 15le 2 Kb - Expected bands Observed Bands Restriction # of Band size(s) # of Band size(s) E e bands b Bands b Bam HI 1 >2.0 l 9.1 Eco RI 3 0.35, >07, >09 3 0.35, 1.5, 4.0 Hind HI 1 >2.0 1 5.5 XbaI 2 >07, >13 2 5.0, 7.5 Chapter 3 IN VITRO AND WHOLE PLANT MAGNITUDE AND CROSS RESISTANCE CHARACTERIZATION OF TWO MIDAZOLINONE-RESISTAN T SUGARBEET (Beta vulgaris) SOMATIC CELL SELECTIONS 139 140 In Vitro and Whole Plant Magnitude and Cross Resistance Characterization of Two Imidazolinone-Resistant Sugarbeet (Beta vulgaris) Somatic Cell Selectionsl TERRY R. WRIGHT and DONALD PENNER’ Abstract. ALS-inhibiting herbicide carryover in soil can severely afi‘ect sugarbeets grown in the year(s) following application. Two newly developed imidazolinone-resistant (IMI- R) sugarbeet somatic cell selections (Sir-13 and 93R3 OB) were examined for magnitude of resistance and extent of cross resistance to other classes of ALS-inhibitors and compared to a previously developed sulfonylurea-resistant (SU-R) selection, Sur. In vitro shoot culture tests indicated Sir-13 resistance was specific to imidazolinone (IMI) herbicides at approximately a 100-fold resistance as compared to the sensitive control sugarbeet. Sur was 10000-fold resistant to the sulfonylurea (SU) herbicide, chlorsulfirron, and 40-fold resistant to the triazolopyrimidine sulfonanilide (TP) herbicide, flumetsulam, but not cross resistant to the IMI herbicides. 93R30B was selected for IMI-R from a plant homozygous for the SU—R allele, Sur, and displayed similar in vitro SU-R and TP-R as Sur, but also displayed a very high resistance to various IMI herbicides (400- to 3600-fold). Compared to the sensitive control, Sir-13 300- and >250-fold resistant to imazethapyr and imazamox residues in soil, respectively. Response by whole plants to POST herbicide applications was similar to that seen in shoot cultures. Sir-13 exhibited >100-fold resistance to ' Received for publication ............. ,1997, and in revised form ............. , 1997. 2 Grad. Res. Asst. and Prof, Dep. of Crop and Soil Sci., Michigan State Univ., East Lansing, MI 48824- 1325. 141 imazethapyr as well as imazamox, and 93R30B showed >250-fold resistance to both herbicides. 93R3 OB showed great enough resistance to imazamox to merit consideration of imazamox for use as a herbicide in these beets. Sir-l3 showed a two- to three-fold higher level of resistance in the homozygous versus heterozygous state, indicating that like most ALS-inhibitor resistance traits, it was semidominantly inherited. Nomenclature: chlorsulfirron, 2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2- yl)amino]carbonyl]benzenesulfonamide; flumetsulam, N-(2,6-difluorophenyl)-5- methyl[1,2,4]triazolo[1,5a]pyrimidine-2-sulfonamide; imazamox, 2-(4-isopropyl-4-methyl- 5-oxo-2-irnidazolin-2-yl)-5-(methoxymethyl) nicotinic acid; imazaquin, 2-[4,5-dihydro-4- methyl-4-(l-methylethyl)-5-oxo-1H-imidazol-2-yl]-3 -quinolinecarboxylic acid; imazethapyr, 2-[4,5-dihydro-4-methyl-4-(l-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl- 3-pyridinecarboxylic acid; sugarbeet, Beta vulgaris L. ‘EL-49’, ‘L03’, and ‘REL—l ’. Additional index words. Acetolactate synthase, AHAS, acetohydroxyacid synthase, Sur, Sir-I3, 93R3OB, sulfonylurea resistance, triazolopyrimidine resistance, somatic cell selection. INTRODUCTION Acetolactate synthase (ALS)3-inhibiting herbicides effectively control a broad spectrum of weeds in corn (Zea mays L.), small grains, and leguminous crops. These 3 Abbreviations: ALS, acetolactate synthase (EC. 4.1.3.18, also known as AHAS, acetohydroxyacid synthase); SU, sulfonylurea; IMI, imidazolinone; TP, triazolopyrimidine sulfonanilide; PTB, pyrimidinylthiobenzoate; SU-R sulfonylurea resistance; IMI-R, imidazolinone resistance; wt, wildtype, herbicide sensitive trait; 150, herbicide concentration or dose required to cause a 50% response effect 142 herbicides kill plants by inhibiting ALS (also called AHAS)’, the first enzyme of the branched chain amino acid (valine, leucine, and isoleucine) biosynthetic pathway. Classes of herbicide chemistry that act by inhibiting the ALS enzyme include: the sulfonylureas (8103, the imidazolinones (IMI)3, the triazolopyrimidine sulfonanilides (TP)3, and the pyrimidinylthiobenzoates (PTB)3 . The ALS-inhibiting herbicides have gained wide acceptance due to their low use rates, low mammalian toxicity, and the wide range of weeds controlled. Residual herbicide activity can be extremely beneficial for tolerant crops by preventing weed competition throughout the growing season; however, persistence of herbicides in the soil can injure susceptible rotational crops (Renner and Powell 1991; Walsh et al. 1993; Johnson et al. 1993; Krausz et al. 1994). Sugarbeets are sensitive to most ALS-inhibiting herbicide residues but carryover injury from IMI herbicides is greater than with other classes (Moyer et al. 1991). Label restrictions prevent sugarbeets from being planted for the next 18‘, 26’, and 406 months in fields where primisulfuron (an SU), flumetsulam (a TP), or imazethapyr (an IMI), respectively, have been applied. IMI and SU herbicide persistence is significantly affected by soil and environmental factors, making the risk of herbicide carryover injury difficult to predict (Frederickson and Shea 1986; Stougaard et al. 1990). Weed control in sugarbeets is also notoriously costly and laborious, ofien requiring multiple herbicide applications and versus the nontreated control; R/S, resistance ratio: the 150 of the resistant line divided by the sensitive, wt 150; CMS, cytoplasmically male sterile, ‘ BeaconD Herbicide label, Ciba, Research Triangle Park, NC. ‘ Broadstrike" Herbicide label, DowElanco, Indianapolis, IN. 143 cultivations due to the diversity of weeds infesting sugarbeets, the slow and non- aggressive growth of sugarbeets, and marginal sugarbeet tolerance to most herbicides (Winter and Wiese 1982; Schweizer and Dexter, 1987; Shribbs et al. 1990). Development of sugarbeets resistant to ALS-inhibiting herbicides could minimize the potential for herbicide carryover injury, Shorten rotation restrictions for sugarbeets, increase flexibility for crops grown in rotation with sugarbeets, and potentially provide new, efl‘ective weed control options for sugarbeets. Sugarbeet variants resistant to selected SU herbicides (Saunders et al. 1992; Hart et al. 1992, 1993), IMI and IMI plus SU herbicides (Wright and Penner 1997) (Sur, Sir-13, and 93R30B, respectively) were developed from tissue culture by somatic cell selection. SU- resistance (SU-R)3 was due to an ALS enzyme with reduced sensitivity to specific SU herbicides and inherited as a semidominant allele called Sur (Saunders et al. 1992; Hart et al. 1993). IMI-specific resistance (IMI-R)3 and broad cross resistance to IMI and SU herbicides (IMI-R/SU-R) were inherited as the monogenic semidominant alleles, Sir13 and 93R3OB, respectively (Wright and Penner 1997). All three resistance traits were allelic. Many other ALS-based resistances are inherited as semidominant traits (Chalelf et al. 1984; Anderson and Georgeson 1989; Sebastian et al. 1989; Haughn et al. 1990). A semidominant resistance trait expresses a higher level of resistance in the homozygous versus the heterozygous condition. Unfortunately, sugarbeets have only one ALS locus (Hartnett et al. 1991; Wright and Penner 1997) allowing only one allele to be expressed in the homozygous state. This would impede traditional breeding attempts to combine the 6 Pursuito Herbicide label, American cyanamid Company, Wayne, NJ. 144 class-specific resistances of Sur and Sir-l3, since both alleles could only exist in the heterozygous condition in the same plant. The potential to increase the spectrum of resistance may be facilitated by the 93R3OB allele, resistant to IMI and SU herbicides. The objectives of the following studies were to determine the magnitude of imidazolinone resistance and of cross resistance by sugarbeet variants to various ALS- inhibiting herbicides fi'om difl‘erent chemistry classes. MATERIALS AND METHODS In vim shoot culture resistance. Clonal shoot cultures containing the three respective herbicide-resistant sugarbeet selections (Sur, Sir-l3, and 93R3 OB) and one sensitive wildtype (wt)3 sugarbeet clone (REL-1) 7 were repeatedly multiplied in vitro to increase shoot numbers for cross resistance screening at the regenerate generation (Rn) level. Sur and Sir-13 R0 shoot clones were derived from REL-1 cultures by acute selection with chlorsulfuron and imazethapyr, respectively (Saunders et al. 1992; Wright and Penner 1997). REL-l exhibited sensitivity to all ALS-inhibiting herbicides tested and served as the sensitive control in resistance characterization experiments. 93R30B was derived from a Sur descendant, 93R307 (chosen for its amenability to tissue culture selection and plant regeneration and for homozygosity for the Sur trait), by acute selection on imazethapyr (Wright and Penner 1997). Shoot cultures were initially assessed in vitro for herbicide magnitude and cross resistance by measuring their response to logarithmically increasing herbicide concentrations. Three uniform, freshly-subdivided shoots were placed onto M20 media 145 (Wright and Penner 1997) supplemented with lO-fold increasing concentrations of ALS- inhibiting herbicides ranging from 1 nM to 100 nM. Response to three IMI herbicides (imazamox, imazaquin, and imazethapyr), one SU herbicide (chlorsulfirron), and one TP herbicide (flumetsulam) was determined. Shoot cultures were maintained at 25 C under 20 uEm’zs'l continuous fluorescent lighting for 3 weeks. Shoot injury was rated and shoot fresh weights per plate determined to assess culture responses to various herbicides. Each experiment was conducted twice as a completely randomized design with four replications per treatment. Data were combined over experiments. An 150 3 value (herbicide concentration causing 50% injury) was determined for each sugarbeet line with each herbicide by log-logistic analysis (Seefeldt et al. 1995). Resistance ratios (R/S)3 were calculated as the 150 of the resistant line divided by the sensitive, wt 150. Whole plant resistance to simulated imidazolinone herbicide soil residues. Sugarbeet resistance to simulated carryover residues of imidazolinone herbicides was initially screened by PPI herbicide experiments in the greenhouse. Seed for testing 93R3OB in these experiments was not available because 93R3OB was discovered a couple of years after Sir-l3. Sur previously exhibited no resistance to imidazolinone herbicides (Hart et al. 1992 and 1993) and was not included in these experiments. Four sugarbeet lines were tested. F3 seed homozygous for Sir-13 was bulked from four previously identified self— pollinated homozygous F2 plants. F3 seed was also bulked from separately identified homozygous sensitive F2 plants, descendants of the original Sir-13 cross. The sensitive F3 7 Acquired from J. W. Saunders, USDA-ARS, East Lansing, MI. 146 seeds served as an internal sensitive control along with REL-1 S1 seed and a commercial sugarbeet hybrid, ACH-31'. Stepwise increasing rates of imazethapyr and imazamox were applied to an air-dry mineral-based greenhouse substrate9 in 250 nil-plastic pots. Herbicides were applied at 0, 0.3, 1.1, 4.4, 17.5, 70, and 280 g ai ha'l imazethapyr and imazamox in a 239 L ha‘l spray volume with a link belt sprayer. Soil from pots receiving the same herbicide treatment were combined in a 4-L metal cylinder and mixed by tumbling for 30 sec. Treated soil was redistributed to the sprayed pots and four sugarbeet seed balls were planted 1 cm deep in each pot. Pots were maintained in the greenhouse at 23:1:2 C with a 14 h light: 10 h dark photoperiod supplemented with high pressure sodium greenhouse lamps. Injury ratings and g fresh weight per pot were determined 5 weeks after planting. The experiment was conducted twice as a completely randomized design with four replications per treatment and data were combined over experiments. Trends were similar for both methods of measuring plant response; therefore, only fresh biomass reduction will be reported. Rates causing 50% fresh biomass reduction (150) were calculated using the methods of Seefeldt et al. (1995) and corresponding resistance ratios were calculated. Whole plant resistance to POST herbicide applications. Sugarbeet resistance to POST applications of selected imidazolinone and sulfonylurea herbicides was determined by spraying four- to six-leaf sugarbeet plants with stepwise increasing rates of test herbicides. Herbicides were applied as described above at the following rates: 8 American Crystal Sugar Company, 1700 North Eleventh Street, Moorhead, MN 56560. 9 Metro Mix 350, Scotts Inc, 14111 Scottslawn Road Marrysville, OH 40341. 147 imazethapyr10 and imazamoxlo at 0, 0.3, 1.1, 4.4, 17.5, 70, and 280 g ai ha"; chlorsulfuronu at 0, 0.07, 0.3, 1.1, 4.4, 17.5, and 70 g ai ha'l; and sulfometuron-methylll at 0, 0.2, 0.8, 3.1, 12.5, 50, and 200 g ai ha". Herbicide applications were made to: homozygous Sir-13 F3 plants (Sir-13/Sir-13), bulked from nonsegregating F2 progeny, EL-497 sugarbeets (Sur/Sur), REL-l 81 plants (wt/wt), ACH-31 plants (wt/wt), and F2 93R30B plants segregating for the 93R303 allele (i.e., 93R3OB/93R3OB, 93R3OB/Wt, and wt/wt). Sensitive plants within the segregating population were identified and rouged fiom the experiment by using a nondestructive leaf disk assay described by Wright and Penner (1997). The 93RBOB population, therefore consisted of a mixture of homozygous and heterozygous individuals with respect to 93R3OB. True breeding 93R3OB plants were not available for these experiments. Greenhouse conditions were as previously described. Experiments were conducted twice as a completely randomized design with four replications. Data were combined over experiments and 150 rates detemlined for each herbicide and resistance ratios calculated as previously described. Sir-13 allele copy number effect on POST imidazolinone resistance. Quantification of Sir-13 allele copy number effect on whole plant imidazolinone resistance was determined by comparing the response of homogeneous populations of Sir-13/Sir-13, Sir-134w, and wt/lvt to herbicides applied POST. Homozygous Sir-13 populations (Sir-13/Sir-l3) were established from bulked progeny seed of four homozygous F2 plants. Clonal copies of the '0 POST imidazolinone treatments also included 2.4 L ha" SunIt-II (Agsco, PO Box 458, Grand Forks, ND 58206) and 2.4 L ha" 28% N as urea ammonium nitrate). 148 same four homozygous Sir-l3 F2 plants were crossed to cytoplasmically male sterile (CMS)3, herbicide sensitive sugarbeets ‘L03’7 to produce a testcross population homogeneously heterozygous for the Sir-13 trait (Sir-13A"). Homozygous sensitive seed equivalent to the F3 seed above was collected from four self-pollinated, herbicide sensitive F2 plants derived from the original Sir-13 cross. REL-1 S1 plants were also included as a sensitive control. 93R3 OB was not included in these experiments due to low seed availability. Sugarbeets were grown in 250-ml plastic containers in greenhouse substrate at a density of one plant per pot. Plants were sprayed at the four- to six-leaf stage with imazethapyr and imazamox at 0, 0.3, 1.1, 4.4, 17.5, 70, and 280 g ai ha'l as described above. Plants were rated for injury and g fresh weight per pot 3 weeks alter treatment. The study was conducted as a completely randomized design with four replications, repeated, and data combined over experiments. 150 values were determined and resistance ratios calculated for each sugarbeet line as described above. RESULTS AND DISCUSSION In vitro shoot culture resistance. Sugarbeet line, herbicide dose, and line by dose interactions were all significant (P<0.001). Sir-13 and 93R303 shoots exhibited significant resistance to all three IMI herbicides tested but Sur response was not significantly different fiom the wt line, REL-1 (Table 1). The magnitude of resistance to the IMI herbicides varied from 75- to 220-fold for Sir-13, depending on the specific herbicide tested. 93R30B was significantly more resistant to the M herbicides than Sir- 13, displaying resistance levels from 400- to 3600-fold. Sir-13 showed no cross resistance " POST sulfonylurea treatments also included 0.25% v/v le. 149 to the SU herbicide, chlorsulfiiron, nor the TP herbicide, flumetsulam. Sur exhibited a large, 10000-fold level of resistance to chlorsulfilron and a modest 40-fold resistance to flumetsulam. 93R30B, derived from a plant homozygous for Sur, displayed similar levels of resistance to chlorsulfuron and flumetsulam as seen with Sur, but was also highly resistant to the IMI herbicides whereas Sur was sensitive to the IMI herbicides. 93R30B may represent a double mutant ALS allele. Chalefi‘ and Ray (1984) described the selection of two similar SU-R traits (C3 and S4) at two unlinked loci in tobacco plants. In an attempt to increase the level of SU-R, Creason and Chaleff (1988) placed S4S4 cells on a higher level of SU selection. The resulting S4-Hra allele did provide an additional level of SU-R compared to the original S4 allele. Lee et al. (1988) isolated and sequenced the C3 and S4-Hra genes. Both genes contained a change from the highly conserved wildtype proline residue at position 196 of the deduced ALS primary structure. In addition, S4-Hra contained a second mutation, a change from the conserved wildtype tryptophan residue at position 572. Presumably the second mutation afl‘orded S4-Hra the elevated SU resistance level versus C3 and S4. Mutations at the analogous proline site have been observed in SU-R variants of Arabidopsis thaliana L. (Haughn et al. 1988), prickly lettuce (Lactuca serriola L.), and kochia (Kochia scoparia (L.) Schrad.) (Guttieri et al. 1992). Analogous mutations to the change at only the tryptophan residue have been Observed to provide a very high level of SU-R as well as resistance to all other classes of ALS-inhibitor herbicides in several plant species (Bemasconi et al. 1995; Woodworth et al. 1996). Creason and Chaleff (1988) did not report any tests for cross resistance. Broad cross resistance has also been achieved by intragenic recombination of respective, (IMI and PTB)-specific (Csr1-2 allele) and (SU 150 and TP)-specific(Csr1-l allele) mutations to create a recombinant double mutant (Csr1-4 allele) resistant to all four classes (Mourad et al. 1992, 1994). Wright and Penner (1997) indicated the Sur, Sir-13, and 93R3OB alleles appear to reside at the single ALS locus in sugarbeets. Sur SU-R specificity is similar to the Csrl -1 allele in Arabidopsis (Haughn and Sommerville, 1986; Mourad et al. 1992) and Sir-l3 IMI-R specificity is similar to the Cer-2 allele in Arabidopsis (Haughn and Sommerville 1990; Mourad et al. 1992) or as seen in corn and cocklebur (Xanthium strumarium L.) (Bemasconi et al. 1995). The level of IMI-R in 93R30B exceeds that observed in Sir-I3 by 5 to 15 times and the level of SU-R and TP-R may be greater (Table l). 93R3OB may represent selection of a double mutant ALS enzyme: either a broad cross resistance mutation (Bemasconi et al. 1995; Woodwoth 1996) or an IMI-specific resistance mutation (Haughn and Sommerville, 1986; Mourad et al. 1992) in addition to the SU-R mutation in Sur. The gene sequence characterization may provide filrther insight into the ALS enzyme binding site changes responsible for the different magnitude and cross resistance characteristics of these three resistance alleles. Whole plant resistance to simulated imidazolinone herbicide soil residues. The rnineral-based greenhouse potting substrate used in these experiments was superior to a true soil system. Preliminary experiments comparing herbicide sensitive sugarbeet response to imazethapyr and imazamox incorporated into potting substrate or field soil (Spinks loamy sand: a sandy, mixed, mesic Psammentic Hapludalfs, pH 6.5, and 1.0% organic matter) indicated herbicide injury to otherwise healthy sugarbeets was similar in either system (data not shown). Using the artificial substrate, however, allowed us to 151 avoid uneven stand establishment due to disease, soil crusting, and the complication of weed competition at the lower herbicide doses. Significant main effects of herbicide rate and sugarbeet type as well as interactions were indicated by the ANOVA (P<0.001). Response of herbicide-sensitive F3 plants, ACH-3 1, and REL-l plants to IMI herbicides did not differ. The homozygous Sir-13 plants displayed a 300-fold level of resistance compared to the wt/wt REL-l plants to imazethapyr (Table 2). All 150 was not reached for imazamox even at the highest rate, 280 g ai ha". The common field use rates for imazamox and imazethapyr are 35 and 70 g ai ha' 1, respectively. Sir-13 resistance was sufficient to tolerate four and five times the field rate of imazethapyr and imazamox soil residues, respectively, and may provide the opportunity to use the herbicides PRE to control weeds directly’in sugarbeets. Whole plant resistance to POST herbicide applications. The main effects and interactions were significant (ANOVA, P<0.001) in the postemergence resistance experiments. Sir-13, as in the in vitro studies, showed around 100-fold resistance to both IMI herbicides, but no resistance to the two SU herbicides tested (Table 3). Sur displayed a greater than 1000-fold level of resistance to chlorsulfuron, but much more modest 20- fold level to sulfometuron. This is consistent with previous Observations, that Sur is resistant to selected members of the SU class of chemistry (Hart et al. 1992). A measurable and significant level of resistance to the IMI herbicides was shown by Sur; however, the three- to four-fold resistance has little utility in cropping situations. Other SU class-specific variants have also showed similar low levels of resistance to the IMI herbicides (Haughn et al. 1986, Mourad et al. 1992). 93R30B was highly resistant to both M herbicides applied POST, displaying 300- and >250-fold resistance to 152 imazethapyr and imazamox, respectively (Table 3). Response to the SU herbicides was significantly different fiom the wt sugarbeet REL-1 but much lower resistances were observed than seen with the Sur trait. This seemed to contradict the data of Table 1 showing 93R30B equally resistant to chlorsulfuron as Sur; however, in this experiment, 93R30B plants were from a segregating F2 population, thus also incorporating the sensitive allele into the population. A reduced whole-plant level of resistance would then be expected if 93R3OB is a semidominant trait as are most other ALS-based resistances (Anderson and Georgeson, 1989; Sebastian, 1989; and Haughn et al. 1990). Additionally, in Table 1, the “93R30B ” represents the original regenerate fi'om tissue culture meaning the genotype of this regenerate was 9R3OB/Sur. One half of the ALS enzyme activity in the R0 generation was of Sur origin which would mask any potential differences between Sur and 93R30B in vitro. POST application of imazethapyr to Sir-13 would not be recoMended, even though a 100-fold resistance is seen. Sugarbeets are so sensitive to this herbicide, that an unacceptable level of injury still would occur. POST application of a field rate of imazamox to Sir-13, or imazethapyr to 93R3OB, may be possible; however, the level of protection was marginal for these IMI herbicides at field rates when tested in the greenhouse. All 150 was never reached for 93R3OB with imazamox applied up 280 g ai ha’ I, an 8X field rate. It appears that POST application of imazamox for weed control is feasible with 93R30B. The potential exists to avoid‘sugarbeet injury and yield losses caused by current chemical weed control practices as observed by Dexter (1994) and Starke and Renner (1996). By creating a high margin of crop safety to herbicides that control the same or broader weed spectrum, sugarbeet yields may be increased. 153 Sin-I3 allele copy number effect on POST imidazolinone resistance. Resistance to ALS-inhibiting herbicides has typically been inherited as a semidominant trait. Plants in the homozygous condition have been more resistant than heterozygotes to the same herbicide (Chaleff and Ray 1984; Anderson and Georgeson 1989; Sebastian et al. 1989; Mallory-Smith et al. 1990). A similar trend is seen in IMI-R in the Sir-13 trait. Sir-13 in the heterozygous state afi‘orded plants 45- and 62-fold resistance to imazamox and imazethapyr, respectively, versus the tht control. Sir-13 resistance to POST IMI in the homozygous state was two- to three-fold higher than in the heterozygous state (Table 4). On the other hand, the sensitive F3 counterpart to the Sir-13 homozygote showed no difl‘erence in herbicide tolerance versus the REL-1 sensitive line. These data would indicate that the greatest margin of safety can be met by maintaining ALS-resistance traits in the homozygous condition. With the invention of IM-R and IMI-R/SU-R sugarbeets, the potential to avoid IMI and SU herbicide carryover injury to sugarbeets may soon be realized. This could potentially reduce rotation restrictions for planting Sir-l3 and 93R30B sugarbeets in fields with ALS-inhibiting herbicide residues, and increase the flexibility growers have in their cropping rotation. Additionally, the level of imazamox resistance in 93R3OB appears high enough to consider using this herbicide POST for weed control in sugarbeets. ACKNOWLEDGEMENTS The authors express their appreciation to Dr. Joseph Saunders for his technical assistance and advice in developing and breeding these sugarbeets and to Wendy Pline, 154 Beth McNielly, and Amy Gonzalez for assistance with tissue culture experiments. This research was funded by American Cyanamid. 155 LITERATURE CITED . Anderson, P. C. and M. Georgeson. 1989. Herbicide-tolerant mutants of corn. Genome 31:994-999. . Bemasconi, P, A R. Woodworth, B. A Rosen, M. V. Subramanian, and D. L. Siehl. 1995. A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase. J. Biol. Chem. 270:17381-17385. . Chalefl‘; R S. and T. B. Ray. 1984. Herbicide-resistant mutants from tobacco cell cultures. Science 223:1148-1151. . Creason, G. L. and R S. Chalefl‘. 1988. A second mutation enhances resistance of a tobacco mutant to sulfonylurea herbicides. Theor. Appl. Genet. 76: 177-182. . Dexter, A. G. 1994. History of sugarbeet (Beta vulgaris) herbicide rate reduction in North Dakota and Minnesota. Weed Technol. 82334-337. . Frederickson, D. R. and P. J. Shea. 1986. Effect of soil pH on degradation, movement, and plant uptake of chlorsulfiiron. Weed Sci. 34:328-332. . Guttieri, M. J., C. V. Eberlein, C. A. Mallory-Smith, D. C. Thill, and D. L. Hoffman. 1992. DNA sequence variation in Domain A of the acetolactate synthase genes of herbicide-resistant and -susceptible weed biotypes. Weed Sci. 402670-676. . Hart, S. E., J. W. Saunders, and D. Penner. 1992. Chlorsulfuron-resistant sugarbeet: cross resistance and physiological basis of resistance. Weed Sci. 402378-383. 156 9. Hart, 8. E., J. W. Saunders, and D. Penner. 1993. Semidominant nature of 10. 11. 12. 13. 14. 15. monogenic sulfonylurea herbicide resistance in sugarbeet (Beta vulgaris). Weed Sci. 41:317-324. Hartnett, M. E., C.-F. Chui, S. C. Falco, S. Knowlton, C. J. Mauvais, and B. J. Mazur. 1991. Molecular characterization of sulfonylurea resistant ALS genes. Pages 343-353 in J. C. Caseley, G. W. Cussans, and R. K. Atkin, eds. Herbicide Resistance in Weeds and Crops. Butterworth-Heneman, Ltd., Oxford, England. Haughn, G. W. and C. Sommerville. 1986. Sulfonylurea-resistant mutants of Arabidopsis thaliana. Mol. Gen. Genet. 204:430-434. Haughn, G. W., J. Smith, B. Mazur, and C. Somerville. 1988. Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides. Mol. Gen. Genet. 2112266-271. Haughn, G. W. and C. R. Somerville. 1990. A mutation causing imidazolinone resistance maps to the Cer locus ofArabidopsis thaliana. Plant Physiol. 9221081-1085. Johnson, D. H. and R. E. Talbert. 1993. Imazaquin, chlorimuron, and fomesafen may injure rotational vegetables and sunflower (Helianthus annuus). Weed Technol. 72573-577. Krausz, R. F., G. Kapusta, and J. L. Matthews. 1994. Soybean (Glycine max) and rotational crop response to PPI chlorimuron, clomazone, imazaquin, and imazethapyr. Weed Technol. 82224-230. 16. 17. 18. 19. 20. 21. 22. 23. 157 Lee, K. Y., J. Townsend, J. Tapperman, M. Black, C.-F. Chui, B. Mazur, P. Dunsmuir, and J. Bedbrook. 1988. The molecular basis of sulfonylurea herbicide resistance in tobacco. EMBO J. 721241-1248. Mallory-Smith, C. A., D. C. Thill, M. J. Dial, and R. S. Zemetra. 1990. Inheritance of sulfonylurea herbicide resistance in Lactuca spp. Weed Technol. 42787-790. Mourad, G. and J. King. 1992. Effect of four classes of herbicides on grth and acetolactate-synthase activity in several variants ofArabidopsis thaliana. Planta 188:491-497. Mourad, G., G. Haughn, and J. King. 1994. Intragenic recombination in the CSRl locus ofArabidopsis. Mol. Gen. Genet. 2432178-184. Moyer, J. R, R. Esau, and G. C. Kozub. 1990. Chlorsulfuron persistence and response of nine rotational crops in alkaline soils of southern Alberta. Weed Technol. 4:543-548. Renner, K. A. and G. E. Powell. 1991. Response of sugarbeet (Beta vulgaris) to herbicide residues in soil. Weed Technol. 52622-627. Saunders, J. W., G. Acquaah, K. A. Renner, and W. P. Doley. 1992. Monogenic dominant sulfonylurea resistance in sugarbeet from somatic cell selection. Crop Sci. 32:1357-1360. Scweizer, E. E. and A. G. Dexter. 1987. Weed control in sugarbeets (Beta vulgaris) in North America. Rev. Weed Sci. 32113-133. 24. 25. 26. 27. 28. 158 Sebastian, S. A., G. M. Fader, J. F. Ulrich, D. R. Fomey, and R S. Chaleff. 1989. Semidominant soybean mutation for resistance to sulfonylurea herbicides. Crop Sci. 2921403-1408. Seefeldt, S. S., J. E. Jensen, and E. P. Fuerst. 1995. Log-logistic analysis of herbicide dose-response relationships. Weed Technol. 92218-227. Shribbs, S. M., D. W. LyBecker, and E. E. Schweitzer. 1990. Bioeconomic weed management models for sugarbeet (Beta vulgaris) production. Weed Sci. 38:436-444. Starke, R. J. and K. A. Renner. 1996. Velvetleaf (Abutilon theophrasti) and sugarbeet (Beta vulgaris) response to triflusulfuron and desmedipham plus phenmedipham. Weed Technol. 102121-126. Stougaard, R. N., P. J. Shea, and AR Martin. 1990. Effect of soil type and pH on adsorption, mobility, and efficacy of imazaquin and imazethapyr. Weed Sci. 38:67-73. 29. Welsh, J. D., M. S. Defelice, and B. D. Sims. 1993. Soybean (Glycine max) herbicide carryover to grain and fiber crops. Weed Technol. 72625-632. 30. Winter, S. R. and A. F. Wiese. 1982. Economical control of weeds in sugarbeets 31. (Beta vulgaris). Weed Sci. 30:620-623. Woodworth, A. R, B. A. Rosen, and P. Bemasconi. 1996. Broad range resistance to herbicides targeting acetolactate synthase (ALS) in a field isolate OfAmaranthus sp. is conferred by a Trp to Leu mutation in the ALS gene. Plant Physiol. 11121353. 159 32. Wright, T. R. and D. Penner. 1997. Selection and genetic analysis of two imidazolinone-resistant sugarbeets. Abstr. Weed Sci. Soc. Amer. 37:253. 160 BEEF—om co: E 82053: e838; acme—388 3053mm < .Amodndv begomiwfi SEE 8: ea 20383350 3 88:2: 3:0 5:38 02038: a 553$ Cozu— 088 of .3 3338 Sun— a .3 cube: 74mm 03228 05 3 Eat? 8832 :08 no a— we owe 35368 n mg a 5%.: 80% $3 8:8 2 @8362 5:82.083 02053: 2:. x 8 e 8: x 82: n 8.5 x 83 o 828 x e9. o 88. x 82 o 88a momma x9. 25: x82: 288 82 am e: as. c2 2: 5m x _ a om x _ a 9m x as e 83 x me e 82 x ea e 82 new e um a 2 a a: a 8 .8 em as 93 93 . 93 93 93 m2 3 ma 3 we 3 m2 3 a ma . 3 82o session EC 5.3385 Se 8:53on 5,5 33.885 :2: 533:: :2: 8.585 $283.5: wfifififiéé 32:3 3 3:332 380 v5 beam—awn:— BBBo 82m .~ 8335 161 Table 2. Response of Sir-13/Sir-13 F3 plants to simulated (PPI) imidazolinone carryover residues. wt/ivt' Sir-13/Sir-13 Herbicide 1,0b 150 R/S° (g ai ha“) (g ai ha") Imazamox 2.5 >230 >110 xd Imazethapyr 0.8 240 300 x‘ ' Corresponding sensitive Sir- I 3 F3 seed (progeny of homozygous sensitive F2 mother plants) and commercial hybrid ACH-31 responses did not differ significantly from wildtype REL-l 81 plants (wtwt). b The herbicide concentration required to cause 50% shoot fresh weight reduction versus no-herbicide controls. ° R/S = resistance ratio of 150 of each resistant variant to the sensitive REL-1 wildtype 150. d Resistant line 150 is significantly greater than the sensitive 150 (a=0.05). 162 0000000 000 003 00500000 0303 000.5 50000. $3 0003 00:30 0000 500—3: 05 005 088% 00 0050: mm 020> _. 00550000 000 0 00203000 0002500 00309000 3050503 < .Godfldv 200005500 00.56 .00 00 000505000000 3 050065 200 08200 0203000 0 02533 0050— 088 05 3 330:8 0009 a .3 0.50:3 Tqmm 0300.000 05 05 00050> 00050500 0000 00 3 .00 0500 00005.50. n m2 .. 205000 0203000 00 000000, 00502000 0303 0005 500% Ram 0300 05 0050000 00505000000 020300; 05. x q a 3 x a a 2 x 08 o o: x 3% 0 .8? 0928 030 um xSSA 32A 02 $0 03 a 3 3m 02 a no 0: ~80 082 £0 08: 3: 2-00 a No 0 8.0 0 no 00 2 E 9.2 0 3 A72. 0 8 0.2 0 00 £2 0 3 m2 3 m2 3 ma 3 ._ ma . 3 2.: .8033 53 000308055 5% 005530020 925 $0050008~ £35 00830:: 33.02. am 05 0.0 8:00-500 2 389.2 080003. 000 223 .0 02.0 163 < 00000000 000 0_ 00205000 000300 0000000000 .00000000 Amodudv €000E0m0 00.000 000 00 0000000000000 3 0000005 300 000000 02000000 0 5003 0000— 2000 000 00 0032.00 00D 0 .3 003003 749— 0300000 000 00 00000> 00000000 0000 00 30 00 0000 000000000 u m3 .0 000.0000 02000000 00 00000> 00000000 00303 0000 00000 $00 00000 00 0000000 00000000000 0203000 00 0. x 8. 0 00 x 00 0 a x _ 0 0.0 0 0.0 000000000 032 3: x a. £00 0: 0 2 .0 Z 5055 0.2 0 00 0.2 0 00 0.2 0 00 0.0. 0 00 0R 3 0R 3 0R 2 .. 02 . a. 02200: 5.005.000 0.02.000 0300 030.0 N 0 o 300 000050 0000 20:0 0.70.0 0000000000 00000000200 .500 00 0000000000 00 000050 0000 0.000 2.0.0. 00 800.5 .0 030.0 Chapter 4 BIOCHEMICAL MECHANISM AND MOLECULAR BASIS FOR ALS-INHIBITING HERBICIDE RESISTANCE IN SUGARBEET (Beta vulgaris) SOMATIC CELL SELECTIONS 164 165 Biochemical Mechanism and Molecular Basis for ALS-Inhibiting Herbicide Resistance in Sugarbeet (Beta vulgaris) Somatic Cell Selections1 TERRY R. WRIGHT and DONALD PENNER’ Abstract. Three sugarbeet selections differing in cross resistance to three ALS-inhibiting herbicide chemistry classes have been developed by somatic cell selection. Sugarbeet selections resistant to imidazolinone herbicides, Sir-13 and 93R30B, do not metabolize ["C]-imazethapyr any faster or differently than sensitive, wildtype sugarbeets or a sulfonylurea-resistant/miidazolinone-sensitive selection, Sur. ALS enzyme extract specific activity from the three herbicide resistant selections ranged from 73 to 93% of the wildtype enzyme extracts in the absence of herbicide, indicating enzyme overexpression was not a factor in resistance. ALS activity extracted fi'om Sir-13 plants showed a 40-fold resistance to imazethapyr but no resistance to chlorsulfuron or flumetsulam. PCR amplification and sequencing of two regions of the ALS gene spanning all known sites for ALS-based herbicide resistance in plants indicated a single nucleotide change in the Sir-13 gene (G337 to A337) resulting in a deduced substitution of threonine for alanine at position 113 in the sugarbeet amino acid sequence. Sur ALS extract activity was not significantly resistant to imazethapyr but were 1000- and SO-fold resistant to chlorsulfuron and flumetsulam, respectively. Sur gene sequencing indicated a single nucleotide change (C562 ‘ Received for publication .............. 1997, and in revised form ............. , 1997. 2 Grad. Res. Asst. and Prof., Dep. of Crop and Soil Sci., Michigan State Univ., East Lansing, MI 48824-1325. 166 to T352) resulting in a serine for proline substitution at position 188 of the ALS primary structure. The 93R3OB nucleotide sequence indicated two mutations resulting in two deduced amino acid substitutions: threonine for alanine at position 113 plus serine for proline at position 188. The 93R3OB double mutant incorporated the changes observed in each of the single mutants above and correlated with higher resistance levels to imazethapyr(>1000-fold), chlorsulfuron (43 00-fold), and flumetsulam (ZOO-fold) at the ALS enzyme level than observed in either of the single mutants. 93R3OB represents the first double mutant derived by a two step selection process which incorporates two class- specific ALS-inhibitor resistance mutations to form a single broad cross resistance trait. The interaction of the two altered amino acids is synergistic with respect to enzyme resistance versus the resistance afforded by each of the individual mutations. Nomenclature: chlorsulfiiron, 2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin—2- yl)amino]carbonyl]benzenesulfonamide; flumetsulam, N-(2,6-difluorophenyl)-5- methyl[l,2,4]triazolo[1,5a]pyrimidine-2-sulfonamide; imazethapyr, 2-[4,5-dihydro-4- methyl-4-(l-methylethyl)-5-oxo-1H—imidazol-Z-yl]-5-ethyl-3-pyn'dinecarboxylic acid; sugarbeet, Beta vulgaris ‘ACH-31’, ‘EL-49’, and ‘REL-l’. Additional index words. Acetolactate synthase, AHAS, acetohydroxyacid synthase, Sur, Sir-13, 93R3OB, imidazolinone resistance, somatic cell selection, sulfonylurea resistance, triazolopyrimidine resistance. 167 INTRODUCTION Acetolactate synthase (ALS)3 is the first enzyme in the biosynthetic pathway of the branched chain amino acids: valine, leucine, and isoleucine (Umbarger, 1978). Four classes of herbicides inhibit this enzyme, including the imidazolinones (IMI)3 (Shaner et al. 1984), the sulfonylureas (SU)3 (Ray 1984), the triazolopyrimidine sulfonanilides (m3 (Gerwick et al. 1990), and the pyrimidinylthiobenzoates (PTB)3 (T akahashi et al. 1991). These herbicides have gained wide acceptance for their low application rates, broad spectrum of weeds controlled season-long, and low mammalian toxicity (Saari et al. 1994). Secondary weed flushes can be controlled by the persistence of herbicide residues in the soil during the growing season. While herbicide persistence is beneficial for weed control in tolerant crops, herbicide carryover injury can result when herbicide residues persist at high enough concentrations to affect sensitive rotational crops (Renner and Powell 1991; Walsh et al. 1993; Johnson et al. 1993; Krausz et al. 1994). Some crops like sugarbeets and lentils (Lens esculenta L.) are extremely sensitive to ALS-inhibiting herbicide carryover (Renner and Powell 1991; Moyer et al. 1991). The development of crops resistant to ALS-inhibitors have the potential to overcome carryover injury risks to the crop and possibly provide a novel crop selectivity allowing the use of proven 3 Abbreviations: ALS, acetolactate synthase (EC. 4.1.3.18, also known as AHAS, acetohydroxyacid synthase); SU, sulfonylurea; IMI, imidazolinone; TP, triazolopyrimidine sulfommilide; PTB, pyrimidinylthiobenzoate; SU—K sulfonylurea resistant; SU-S, sulfonylurea sensitive; IMI-S, imidazolinone sensitive; IMI-IL imidazolinone resistant; PCR, polymerase chain reaction; TLC, thin layer chromatography; wt, wildtype, herbicide sensitive allele; FAD, flavin adenine dinucleotide; 150, herbicide 168 herbicides directly in crops normally sensitive to the herbicide. Crops resistant to ALS- inhibiting herbicides have been derived by somatic cell selection (Anderson and Georgeson 1989; Saunders et al. 1992; Heering et al. 1992; Iler et al. 1993), seed mutagensis (Sebastian et al. 1989; Newhouse et al. 1992), plant transformation (Haughn et a1. 1988; McHughen 1989; D’Halluin et al. 1992), and interspecific crosses (Mallory-Smith et al. 1990). Three sugarbeet alleles encoding resistance to various ALS-inhibiting herbicides were recovered by tissue culture somatic cell selection. A sulfonylurea-resistant (SU-R)3 sugarbeet, Sur, was developed from a SU-sensitive (SU-S)3 sugarbeet clone, ‘REL-l ’, by in vitro cell selection on chlorsulfiiron (Saunders et al. 1992) The SU-R trait, Sur, was semidominantly inherited and provided resistance to specific SU herbicides and the TP herbicide, flumetsulam, but no resistance to the [MI herbicides (Hart et al. 1992, 1993; Wright and Penner 1997). An IMI-specific resistant sugarbeet selection, Sir-13, was developed from the IMI-sensitive (IMI-S)3 clone, REL-l, by selection on imazethapyr (Wright and Penner 1997). The IMI-resistance (IMI-R)3 trait, Sir-13, was also semidominantly inherited (Wright and Penner 1997). A sugarbeet allele, 93R3OB, affording resistance to IMI, SU, and TP herbicides was derived from a IMI-S/SU-R/TP—R sugarbeet (homozygous for Sur) by cell selection on imazethapyr (Wright and Penner 1997). Sur, Sir-13, and 93R3OB are allelic and (semi)dominantly inherited (Wright and Penner 1997). The basis of SU-R in Sur was shown to be an altered sensitivity of the ALS enzyme to specific SU herbicides (Hart et al. 1992). Since all three resistance traits in concentration causing 50% reduction in enzyme activity; R/S, resistance ratio of test line 150 to wt control 150; CI'P, chloroplast transit peptide; HO-imazethapyr, a-hydroxyethyl-imazethapyr. . 169 sugarbeets are allelic, IMI-R in Sir-13 and 93R3OB were expected to result fiom an altered ALS sensitivity as well. The objectives of the following studies were to examine the sensitivity of ALS enzyme fi'om Sur, Sir-l3, and 93R30B to representative herbicides of the M, SU, and TP herbicides classes. The potential for enhanced IMI metabolism was also investigated. Polymerase chain reaction (PCR)3 technology allowed rapid determination of the molecular lesions in the ALS gene responsible for the observed herbicide resistances in each sugarbeet selection. MATERIALS AND METHODS l‘C—imazethpyr metabolism. Imazethapyr metabolism was investigated as a possible mechanism of imidazolinone resistance in Sir-13 and 93R3OB. Homozygous Sir-13 F3, homozygous Sur ‘EL-49", homozygous sensitive REL-l St, and segregating 93R3OB F2 plants were germinated in the greenhouse in 250-ml plastic pots filled with greenhouse substrates at 2508 C with a 16 h light:8 h dark photoperiod supplemented with high pressure sodium lamps. Sensitive segregates from the 93R3OB F2 population were identified by a non-destructive leaf disk assay described by Wright and Penner (1997) and removed. Plants were thinned to one plant per pot and transferred to a growth chamber with the same conditions. Total leaf area of four-leaf sugarbeet plants was approximated and the corresponding fraction of a 239 L ha“l spray application intercepted was estimated to be 53 ul per plant. ‘ Acquired from J. w. Saunders, USDA-ARS, East Lansing, MI. 170 A ["C]-irnazethapyr spotting solution was prepared to simulate a field POST imazethapyr spray application in both volume and composition (239 L ha'l spray volume + 2.4 L ha" SunIt-II‘ + 2.4 L ha'l 28%N urea ammonium nitrate). The spotting solution was spiked with ring-labeled [“C]-imazethapyr (specific activity 213 kBq mmol'l) at 1.7 kBq per plant (53 ul spotting solution). The spotting solution was applied to the two newest leaves of four-leaf sugarbeet plants in 2- to 3-ul drops using a glass microsyringe. Imazethapyr absorption and metabolism were determined for each sugarbeet line 0, 6, 24, and 96 h after herbicide application. Surface [“C]-imazethapyr (unabsorbed herbicide) from each treated leaf was removed by a 30 sec methanol wash in a 20-ml scintillation vial followed by a brief rinse with a stream of fresh methanol. All wash solutions for each plant were combined and solvent evaporated under nitrogen gas7 at 50 C. Harvested leaves were homogenized in 20 ml methanol for 2 min at one-third maximum settings. Homogenate was clarified by centrifugation for 20 min at 18,000 g. Supernatant (extracted ["C]) was decanted and the pellet was resuspended in 10 ml methanol, centrifiiged as before, and supernatant combined with the first extraction. Methanol was evaporated to dryness under nitrogen gas. 5 BACCTO potting soil, Michigan Peat Co., PO Box 981029, Houston, TX 77098. ‘ strait-11°, AGSCO, Inc, PO Box 458, Grand Forks, ND 58206. ’ N-Evap‘ Analytical Evaporator, Organomation Assoc, Inc., 106 Bear Foot Road, Northborough, MA 01532. ' Virtis “45” ° homogenizer, Virtis Research Equipment, 815 Route 208, Gardiner, NY 12575. 171 Leaf surface wash (unabsorbed ["CD and leaf extracted radioactivity were resuspended in 1 ml methanol. Chlorophyll present in leaf extracts efi‘ectively quenched liquid scintillation quantification of extracted radioactivity. Pigments were oxidized by adding 100 pl commercial bleach (5.25% w/v NaOCl) to 100 pl extract aliquots. Recovered radioactivity was determined by liquid scintillation spectroscopy in 15 ml scintillation cocktailg. Measurements were made in duplicate and averaged. Metabolism of absorbed [“C]-imazethapyr was determined by thin layer chromatography (TLC)4 as described by Tecle et al. (1993). Prechanneled TLC plates10 were spotted with 0.17 kBq aliquots of each leaf extract in duplicate. Unlabeled parent imazethapyr (100 rag) and authentic, unlabeled a-hydroxyethyl-imazethapyr (AC 266,511) (100 ug) were also included to identify potential metabolites by co-migration. TLC plates were allowed to completely dry before development. Chromotographic separations were conducted using a n-propanolzmethylene chloride2formic acid (5:4:1 v/v/v) solvent system in a pre-equilibrated TLC chamber for 1 h. The solvent front and origin were marked and TLC plates allowed to air dry. Each comer of the chromotography field (bounded by the origin and solvent fiont) were also marked by spotted [”C], allowing visualization of reference points by the TLC plate scanner. Unlabelled standards were visualized by a 15 9 Safety Solve. scintillation cocktail, Research Products International Corp., 410 North Business Center Drive, Mt Prospect, IL 60056. ‘° LD6DF silica gel 60 A analytical TLC plates, Whatrnan Inc., 9 Bridewell Drive Clinon, NJ 07014. 172 min incubation in an iodine vapor chamber. Radioactivity on TLC plates were visualized and quantified by a [“C] plate scanner". The metabolism experiment was conducted as a completely randomized design with three replications per treatment. The experiment was conducted twice with similar results. Data were combined fiom both experiments and AN OVA calculated. ALS enzyme assays. Commonly used procedures were utilized to partially purify ALS from rapidly expanding leaves of greenhouse-grown sugarbeet plants (Hart et al. 1992). Extracts fi'om homozygous Sir-13 and 93R30 F3 plants, homozygous Sur plants ‘EL-49’, and REL-1 Si sensitive wildtype (wt/wt)3 plants were assayed for ALS activity in the presence of 10-fold increasing concentrations of representative herbicides from three classes of ALS-inhibiting herbicides. Sugarbeet plants were grown in 900-ml plastic pots in greenhouse substrate’ in the greenhouse at 25d:3C with a 16 h light:8 h dark photoperiod supplemented by high pressure sodium greenhouse lights. Ten g of the two newest unexpanded leaves from each of several two- to four-leaf sugarbeets of each line were pooled and homogenized in 40 ml cold homogenation buffer [0.1 M K2HP04, pH 7.5, 1 mM Na-pyruvate, 0.5 mM Mng, 0.5 mM thiamine pyrophosphate, 10 M flavin adenine dinucleotide (F AD)3, 10% by vol glycerol] plus 2.5 g polyvinylpolypyrrolidone. The homogenate was filtered through eight layers of cheesecloth and centrifilged at 27,000 g for 20 min. The supernatant was removed and brought to 50% saturation with (NILhSOa. The solution was centrifiiged at 18,000 g for 15 min. The pellet was redissolved in 2 ml resuspension buffer (0.1 M K2HP04, pH 7.5, 20mM Na-pyruvate, and ” Instalmager' plate and gel scanner, Packard Instrument Co., 800 Research Parkway, Meriden, CT 06450. 173 0.5 mM MgC12) and desalted by size exclusion chromatography”. Enzyme extracts were diluted 1:1 (v/v) with resuspension bufi‘er and assayed immediately. ALS activity was measured by adding 0.2 ml of enzyme preparation to 1.3 ml reaction buffer (25 mM K2HP04, pH 7.0, 0.625 mM MgCl2, 25 mM Na-pyruvate, 0.625 mM thiamine pyrophosphate, 1.25 M FAD) and incubated at 35 C for 1 h. Reaction mixtures were supplemented with 10-fold increasing concentrations of representative chemicals fi'om three ALS-inhibitor classes as follows: imidazolinone (0, 50, 500, 5000, 50000, 500000, and 5000000 nM imazethapyr), sulfonylurea (0, l, 10, 100, 1000, 10000, and 100000 nM chlorsulfuron), and triazolopyrimidine sulfonanilide (0, 1, 10, 100, 1000, 10000, and 100000 nM flumetsulam). The reaction was stopped by adding 50 ul 6 N ' H2804 and incubated at 60 C for 15 min. This procedure decarboxylates the ALS enzyme product, acetolactate, to form acetoin (Singh et al. 1988). Acetoin was detected as a colored complex formed after adding lml a—naphthol (25 g L'l) and creatine (2.5g L") in 2.5 N NaOH and incubating 15 min at 60 C (Westerfield 1945). Purchased acetoin was used as a standard for the colorimetric reaction. Acetoin concentrations were determined by measuring the absorption of the reaction solution at 530 nm. Protein concentrations of the extracts were determined by the method of Bradford (1976) using bovine serum albumin for the standard curve. Experiments were conducted as a randomized complete block design. Experiments with each herbicide were conducted twice with three replications per treatment and data were combined over experiments. The concentration of herbicide inhibiting enzyme activity 50% versus the no herbicide control '2 Sephadex G-25 PD-lO column, Pharmacia, Inc., 800 Centennial Avenue, Piscataway, NJ 08854. 174 (150)3 was determined for each sugarbeet line by log-logistic analysis (Seefeldt et al. 1995). The resistance ratio (R/S)3 of resistant to sensitive Isa values was calculated. ALS gene sequencing. Automated sequencing. A polymerase chain reaction approach was utilized to efficiently determined the molecular basis for resistance in each sugarbeet selection. Five highly conserved amino acid residues within the ALS enzyme primary structure have been reported to account for naturally or in vitro selected resistance to ALS-inhibiting herbicides in plants when changed fiom the wildtype amino acid. These residues are depicted in Figure l by the conserved wildtype amino acid listed along a model procALS protein. The chloroplast transit peptide (CTP)3 serves to direct the nuclear-coded, cytosolically translated pro-ALS protein to the chloroplast where the CTP is cleaved to form the mature enzyme (Bedbrook et al. 1995; Bemasconi et al. 1995; Grula et al. 1995). The CTP and its coding region have a much lower degree of homology among ALS enzymes from different species versus the mature enzyme and coding regions. Selected examples of resistant plants and the known molecular changes are listed below the model protein in Figure 1. The five residues are conveniently divided into two groups, labeled Region A and Region B. Wiersma et al. (1989) described subsections (Domains A and B) of these regions which they identify as sulfonylurea resistance sites. Domain A essentially corresponds to a 13 amino acid stretch surrounding the proline site in Region A and Domain B corresponds to a four amino acid stretch at the tryptophan site in Region B. Guttieri et al. (1992) investigated ALS mutations around Domain A by PCR amplification, 175 but did not extend their investigations beyond residues immediately proximal to Domain A Our sequencing strategy was to specifically amplify ALS gene Regions A and B (Figure 1) from genomic DNA isolated from each of the herbicide resistant sugarbeet selections and from wildtype, sensitive sugarbeets. PCR amplification products were sequenced by automated and conventional dideoxynucleotide terminator sequencing. Genomic DNA was isolated fi'om 30 two-leaf plants homozygous for Sur (EL-49), Sir-I3 (F 3 progeny of previously identified F2 homozygotes), and segregating homozygous and heterozygous 93R3OB F2 plants (sensitive F2 plants rouged from segregating population as previously described above). DNA fi'om wildtype (wt/wt) sensitive plants (REL-l and ACH-31‘3) was also extracted using standard techniques (Anonymous 1991). A wildtype sugarbeet ALS gene had been previously isolated and sequenced (Bedbrook et al. 1995)“. Using this sequence, PCR primers were designed to specifically amplify Regions A and B from genomic DNA isolated from each of the sources listed above. PCR primers were synthesized” as follows: ForA = 5 ’CCAATGTGTTTGCTTACCCTGG-3 ’, RevA = 5 ’-CCTTGTCACCTCAACAATTGG—3 ' (Figure 2), ForB = 5 ’-GGTGGAAAATCTCCCAGTTAAG-3 ’, and RevB = 5 ’-CCGATCAATAAGAGGTTCTTCC-3 ’ (Figure 3). ‘3 American Crystal Sugar hybrid 31, 1700 North Eleventh Street, Moorhead, MN 56560. ” Sugarbeet ALS nucleotide sequence obtained from B. Mazur, DuPont Agricultural Products, Wilmington, DE, personal communication. ‘5 PCR primers ordered from Genosys Biotechnologies, Inc, 1442 Lake Front Circle, Suite 185, The Woodlands, TX 77380. 176 A purchased PCR kit16 was utilized to specifically amplify Region A and Region B fiom each sugarbeet line according to protocol from the manufacturer: 2 ug genomic DNA and 200 ng of each forward and reverse primer pair were added to the prescribed buffer system plus 2.5 Units T aq DNA polymerase per PCR reaction. PCR cycle conditions (denature at 95 C for l min, anneal primers at 55 C for 2 min, polymerize DNA at 72 C for 1.5 min) were repeated for 38 cycles after an initial 2 min denaturation step at 95 C. Primer pairs A and B successfillly amplified single bands of the expected Size, 310 and 360 basepairs, respectively, from genomic sugarbeet DNA. PCR fragment populations fi'om each genotype were purified fi'om 1.2% low melt agarose” slab gel according to instructions from the manufacturer”. PCR fragment populations were sequenced using a dye—labeled didieoxynucleotide terminator sequencing kit19 designed for use with automated DNA sequencers. Unincorporated dideoxynucleotide terrninators were removed by spin column separation”. Labeled fragments were evaporated to dryness, resuspended in 4.5 ul of deionized fomamide250nM EDTA, (pH 8.0) (1:5 by vol) and denatured for 2 min at 95 C. Samples were kept on ice until loaded onto a 6% acrylamide (19:1 acrylamidezbis-acrylamide) gel for automated sequence analysis '6 AmpliTaqo DNA polymerase, Perkin-Elmer, 850 Lincoln Center Drive, Foster City, CA 94404. ‘7 Nu-Sieveo low melt agarose, FMC BioProducts, 191 Thornton Street, Rockland, ME 04841. ‘8 Wizardo PCR Preps DNA Purificaton System , Promega Corp., 2800 Woods Hollow Road, Madison, WI 53711. '9 PRISMo Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit, Perkin-Elmer, 850 Lincoln Center Drive, Foster City, CA 94404.. 177 according to instructions of the manufacturer.21 Three PCR fragment populations for each sugarbeet line and Region were independently amplified and sequenced twice on both strands. Manual sequencing. Automated sequence analysis indicated no sequence difi‘erences among any of the sugarbeet lines within Region B. One or more nucleotide changes from the wildtype sequence were observed within Region A for each of the resistant sugarbeet lines. We cloned Region A PCR fragments from Sur, 93R3OB, Sir-13, and wt sugarbeets to facilitate conventional radiolabeled dideoxynucleotide sequencing to confirm nucleotide R0 22cloning vector changes. PCR fragment populations were ligated into the pC according to kit instructions. Cloned fragments were analyzed by methods and materials provided by a manual DNA sequencing kit”. Sequencing reactions were split and applied to a 5% acrylarnide sequencing gel24 at two difi‘erent timings to provide an unambiguous view of the 5 ’ and 3 ’ ends of Region A. Sequencing gel was transferred to blotting paper 2° Centri-Sepo columns, Princeton Separations, Inc., PO Box 130, Adelphia, NJ 07710. 2‘ ABI 373A Automated DNA Sequencer, Perkin-Elmer Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404.. 22 TA Cloning Kito, Invitrogen Corp., 3985 B Sorrento Valley, San Diego, CA 92121. 2’ fmolO DNA sequencing kit, ., 2800 Woods Hollow Road, Madison, WI 53711. 2‘ Long RangerO DNA sequencing gel solution, FMC BioProducts, 191 Thornton Street, Rockland, ME 04841. 178 and dried under heated vacuum for 50 min at 80 C. X-ray film” was exposed to the gel for 2 days and developed. RESULTS AND DISCUSSION “C-imazethapyr metabolism. The effect of time on l‘C-imazethapyr metabolism was significant, but not significance was observed among sugarbeet lines (P=0.05.). No differences were observed in either the rate or amount of l‘C-imazethapyr absorbed. Only the parent and a-hydroxyethyl-imazethapyr (HO-imazethapyr)3 were detected in these experiments. The accumulation of ["C] as the HO-imazethapyr metabolite was directly proportional with the loss of parent imazethapyr over time (Figure 4). Approximately 40% of parent imazethapyr absorbed was hydroxylated 96 h afier treatment. The rate of metabolism did not significantly differ among sugarbeet lines at any time tested. HO-imazethapyr is the initial metabolite in all plant species investigated (Shaner and Mallipudi 1991; Lee et al. 1991; Tecle et al. 1993). Crop tolerance to imazethapyr has been attributed to differential metabolism (Shaner and Mallipudi 1991). Soybeans rapidly convert imazethapyr to the HO-imazethapyr metabolite and subsequently glucosylate the molecule to totally inactivate the herbicide. Some plant species like corn (Zea mays L.) can rapidly metabolize imazethapyr to HO-imazethapyr (Shaner and Mallipudi 1991). Corn does not appreciably metabolize imazethapyr beyond the HO-imazethapyr metabolite. HO-imazethapyr is still active as an ALS-inhibitor; however, it is effectively immobilized in the plant and not transported to the meristem (Shaner and Mallipudi 1991). 2’ Biomax MR0 autoradiography film, 343 State Street, Eastman Kodak Co., Rochester, NY 14650. 179 This explains why corn is normally sensitive to imazethapyr. The rate of conversion to the HO-imazethapyr was more than three-times slower in sugarbeets (half life >96 h) (Figure 4) than in corn (half life=31 h) (Shaner and Mallipudi, 1991). This would allow translocation and accumulation of parent imazethapyr in the meristem and subsequent death to the sugarbeet plant. The slow rate of metabolism in sugarbeets helps to explain the extreme sensitivity beets have to imazethapyr. Enhanced imazethapyr metabolism was not responsible for IMI-R in any of the sugarbeet selections. An IMI-resistant ALS enzyme, however, would allow sugarbeets to continue to metabolize the herbicide at a slow rate and allow the plant to continue to grow, effectively diluting the herbicide within the plant and allowing the plant to survive the herbicide application. ALS enzyme assays. Herbicide dose, sugarbeet line, and interaCtion effects were significant (P<0.001). Trends of ALS sensitivity to representative herbicides from the IMI, SU, and TP herbicide classes were similar to results of in vitro shoot response to the same herbicides (Wright and Penner 1997). Sir-13 ALS enzyme extracts displayed a significant (40-fold) resistance to imazethapyr versus the sensitive wt line (Table l). The 150 for Sir-13 ALS was approximately three-fold higher to chlorsulfuron and flumetsulam than the wt 150; however, these differences were not statistically significant. No cross resistance to SU or TP herbicides was observed in Sir-l3 shoot cultures or whole plants, in concurrence with the ALS enzyme cross resistance tests (Wright and Penner 1996). Sur displayed equal sensitivity to imazethapyr as the wt enzyme. Sur ALS resistance to chlorsulfilron and flumetsulam were 1000- and 50-fold, respectively. These results agreed with the relative level of SU- and TP- resistance observed in shoot culture tests with Sur (Wright and Penner 1997). The cross resistance pattern also confirmed that 180 mutually exclusive, class-specific resistances were expressed by Sir-13 (IMI-R/SU-S/TP- S) and Sur (IMI-S/SU-R/TP-R). Class-specific resistance traits Similar to Sir-13 and Sur have been reported previously (Haughn and Sommerville 1986, 1990; Lee et al. 1988; Newhouse et al. 1991; Bright et al. 1992; Mourad et al. 1992); however, most were considered IMI-specific or SU-specific resistant because the TP herbicides are a relatively new class of herbicides and few of the original herbicide-resistant iSolates were evaluated for sensitivity to 'I'P herbicides. Likewise, PTB (related analogues also have been called pyrimidinyl-oxy-benzoates) are even newer and fewer cross resistance studies have included this chemistry. 93R30B ALS extracts were highly resistant to IMI, SU, and 'IP herbicides. Resistance to imazethapyr (>1000-fold) was more than 25 times higher than the Sir-l3 enzyme resistance (Table 1). 93R308 resistance to chlorsulfilron and flumetsulam was four times greater than the Sur resistance to the same herbicides. These results support the observations of shoot culture cross resistance experiments that showed 93R30B was significantly more resistant to each class of herbicides than either Sur or Sir-13 (Wright and Penner 1997). Whole plant resistance to POST-applied ALS-inhibiting herbicides generally agreed with the enzyme cross resistance data. Sir-l3 was 2100-fold resistant to IMI herbicides but sensitive to SU herbicides. Sur SU-R varied fiom 20- to >1000-fold, depending on the herbicide, but was essentially sensitive to IMI herbicides (Wright and Penner 1996). These results and the lack of enhanced imazethapyr metabolism support altered ALS enzyme sensitivity as the mechanism of resistance for Sir-13. 93R3 OB showed Significant resistance to IMI and SU herbicides at the whole plant level; however, the level of 181 resistance was much lower than expected in light of the great magnitude of resistance observed in shoot culture tests (Wright and Penner 1997) and enzyme extracts (Table 1). Actually, 93R30B plants displayed only about three-fold higher resistance to IMI herbicides than Sir-13 and were more than five-fold less resistant to SU herbicides than Sur. These apparently contradictory data sets may have been caused by testing whole plants that were a segregating homozygous/heterozygous 93R3OB F2 population. The presence of wt alleles could have decreased the population level of resistance. Such a l response would be indicative of a semidominant allele, where trait expression is higher in the homozygous versus heterozygous state. Most altered ALS enzyme resistance traits reported are inherited semidominantly (Haughn and Sommerville 1986,1988; Sebastian et al. 1989; Newhouse et al. 1991), including the sugarbeet herbicide resistance traits Sur (Hart et al. 1993) and Sir-13 (Wright and Penner 1997). Once sufficient quantities of true breeding 93R3OB seed have been established, whole plant resistance studies should be repeated to determine if 93R30B exhibits greater resistance to IMI, SU, and TP herbicides at the whole plant level. Whole plant resistance to ALS herbicides can be explained by a reduced sensitivity of the target enzyme to the herbicides tested. Another potential mechanism, however, could include overproduction of ALS enzyme due to up—regulation (overexpression) of a single gene or gene amplification resulting in increased enzyme levels. Herbicide resistance has been reported through overexpression (Shah et al. 1986; Montoya et al. 1990) and gene amplification (Donn et al. 1984; Hauptmann et al. 1988; Caretto et al. 1994). In either case, the result would increase levels of the wt target enzyme which could bind and temporarily sequester the herbicide in the plant, allowing free enzyme to catalyze its 182 normal reaction unabated. This mechanism was not apparent with Sur, Sir-13, and 93R308 since the ALS specific activity of the wt enzyme extract was greater than or equal to each of the resistant lines (Table 1). The reduced enzyme activity in the resistant lines may be a result of decreased enzyme efficiency due to the same molecular changes imparting herbicide resistance. Detrimental effects on enzyme afinity for its substrate(s) have been reported for Photosystem 11 D1 proteins from triazine-resistant plants (Pfister and Amtzen 1979; Sims Holt et al. 1981; Hart and Stemler 1991) and an engineered glyphosate-resistant 5-enolpyruvylshikimate-3 -phosphate synthase gene (Padgette et al. 1991). Most reports have indicated, however, that ALS herbicide resistant mutants were neutral with respect to ALS substrate affinity or whole plant fitness (Yadev et al. 1986; Newhouse et al. 1991; Holt and Thill 1994; Siehl et al. 1996) but some mutants have shown reduced enzyme activity (Yadev et al. 1986). Mourad et al. (1995) report a change in substrate specificity in the Arabidopsis thaliana (L.) ALS double mutant, Csrl-4, but not in the single mutant ALS enzymes (Cer-1 and Csr1-2). It is also possible that the plant specific activity differences are due to the relative maturity level of leaves used for ALS enzyme extraction and assay. The expression level of ALS varies greatly by the maturity of the source material examined (Keeler et al. 1993). No observations were made in our experiments regarding the possible differences in enzyme source plant maturity for the different lines, but small differences in maturity could allow for the small, measurable difference in ALS specific activity recovered. The resistant sugarbeets may also have gained additional detrimental and undefined mutations at other loci during the selection process. The pleiotropic effect of additional somaclonal variation on ALS expression in vivo is unpredictable. The reason for the small difi‘erences 183 in specific activity recovered for each sugarbeet line remains unclear. Any of the aforementioned factors could account for the activity differences. The establishment of isogenic lines within elite germplasm for each of the resistance traits would allow more controlled comparisons. ALS gene sequencing. Automated sequencing. No differences were observed among three wild type sugarbeets and the three resistant lines Sir-13, Sur, and 93R3OB in Region B of the ALS gene. Single nucleotide changes fi'om the wildtype sequence were observed in Sir-13 and Sur, and two nucleotide changes were detected in 93R3OB within Region A. Each nucleotide change resulted in a deduced amino acid substitution. Each amplified PCR population gave similar results. By removing herbicide sensitive plants from the 93R3OB F2 population, we effectively selected for three-fourths representation of the 93R3OB gene in the PCR population. Sequence analysis clearly indicated two nucleotide changes when analyzed by the automated sequencer. The PCR products for Region A were cloned from two wildtype and three resistant lines to confirm the results by manual sequencing. Manual sequencing. Results of sequencing cloned PCR fragments agreed with the nucleotide changes determined by automated sequencing of PCR fragment populations. Two nucleotide Sites within Region A showed changes from the wildtype sequence. Figure 5 focuses on two sections of the sequencing gel proximal to the changed nucleotide sites. The top panel shows the nucleotide site where a change in Sir-13 versus the wildtype exists (6337 to A337). This mutation changes the deduced amino acid at position 113 of the ALS enzyme primary structure from alanine to threonine. This change corresponds to the more upstream alanine site in Region A (Figure 1). Analogous 184 mutations have been observed in IMI-R/SU-S cocklebur (Xanthium strumarium L.) (Bemasconi et al. 1995) and IMI-R/SU-S corn (Bright et al. 1992)“. No other changes fi'om the wildtype sequence were observed within the examined regions of Sir-13. A C362 to T362 nucleotide transition was observed in Sur (Figure 5, lower panel) coding for a deduced amino acid substitution of serine for proline at position 188. The same amino acid change was seen in IMI-S/SU-R Arabidopsis (Haughn et al. 1988) and different amino acid substitutions seen at the analogous site in IMI-S/SU-R tobacco (Nicotiana tabacum L.) (Lee et al. 1988), prickly lettuce (Lactuca serriola L.), and kochia [Kochia scoparia (L.) Scrad.] (Guttieri et al. 1992). 93R3OB, interestingly, had two nucleotide changes: the combination of the two mutations listed above. Since 93R3OB was selected from a sugarbeet plant homozygous for Sur (called 93R30), the C362 to T362 nucleotide substitution seen in Sur was anticipated. The same mutation acquired in Sir-13 by cell selection on imazethapyr (Gm to A337) also occurred in 93R30B by selection on imazethapyr; however, this time the selection of the double mutant occurred (G337 to A337 plus C362 to T362) yielding two amino acid changes in the ALS enzyme, alanine to threonine at position 113 plus proline to serine at position 188. Three alleles from cell selections for resistance to ALS-inhibiting herbicides have been described. Each selection differed in the magnitude and cross resistance to the IMI, SU, and TP herbicides. A single nucleotide change and corresponding deduced amino acid substitution correlated with each change in the spectrum of herbicide cross resistance. The deduced amino acid changes and corresponding herbicide resistance observed with 2‘ J. A. Greaves, 1C1 Swds, Slater, IA. Personal communication. 185 each mutation are summarized in Figure 6. The whole-plant and ALS enzyme resistance observed for each of the single mutant selections, Sir-13 and Sur, agree with previous changes detected at the analogous sites in other species (Lee et al. 1988; Haughn et al. 1988; Guttieri et a1. 1992; Bright et al. 1992; Bemasconi 1995). The broad cross-resistant double mutant, 93R3OB, was unique because it was derived by a two-step selection of individual mutations which independently provide for selective resistance. A similar two-step selection for low, then high levels of SU resistance in tobacco has been described (Chalefi‘ and Ray 1984; Creason and Chaleff 1988). Lee et al. (1988) reported two amino acid changes in the highly resistant SU-R trait, S4-Hra, shown in Figure 1 as the double mutant proline to alanine plus tryptophan to leucine. S4-Hra was derived from a trait, S4, and expressed about five-fold higher SU-R than S4. Lee et al. (1988) did not report the sequence of the S4 ALS allele, but did indicate the C3 line, having similar SU-R as S4, possessed a glutamine substitution at the proline site indicated in Region A (Figure 1). Presumably, S4-Hra, was derived by selection for the proline to alanine mutation in S4 and gaining the tryptophan to leucine mutation in the second selection process (Figure 1; Lee et al. 1988). Others have shown the tryptophan to leucine mutation alone provides a very high level of resistance to all classes of ALS- inhibitors (Bemasconi 1995; Hattori et al. 1995; Woodworth et al. 1996). Creason and Chaleff (1988) did not report if S4-Hra had gained the cross resistance to other herbicide classes, characteristic of the tryptophan mutation Site. A second ALS double mutant was derived in Arabidopsis by intragenic recombination of two independently-derived ALS resistance traits. A crossover event between ALS allele Cer-I (IMI-S/SU-R) and allele Csrl-Z (IMI-R/SU-S) resulted in allele Csr1-4 186 resistant to all classes of ALS inhibitors (Mourad et al. 1994). Complete characterization of ALS-inhibitor resistance by Csr1-4 gene has not been reported; however, sensitivity to feedback inhibitors valine and leucine was reduced in Cal-4 whereas Cer -1 and Cer-2 feedback sensitivity was unchanged fiom the wildtype allele Csr] allele (Mourad et al. 1995). In a separate effort, an analogous combinaton of the Cer-1 and Csrl-Z mutation sites was achieved by splicing the 5’ end of Cer-1 with the 3’ end of Cer-2 to form a complete Arabidopsis ALS gene incorporating the two mutations involved in class- specific resistances. The chimeric gene conferred cross resistance in transgenic tobacco (Hattori et al. 1992). The exact nature of the combination of these two mutations in the same gene was not well characterized. Our two-step selection of a double mutant, 93R3OB, fiom tissue culture offers a novel approach to derive broad cross resistance to the ALS-inhibiting herbicides. Interestingly the same threonine for alanine substitution derived from a wt (REL-1) gene in Sir-13 was selected as an independent mutation in the SU-R gene, Sur. Indeed, the double mutant was cross resistant to the IMI, SU, and TP herbicides. An unanticipated effect of the double mutation was the synergistic effect with respect to IMI-R observed with in vitro shoots as well as whole plants (Wright and Penner 1996, 1997). Additionally, the 93R30B ALS enzyme was significantly less sensitive to imazethapyr versus Sir-l3 or to chlorsulfuron and flumetsulam versus Sur. The synergistic interaction of the two mutations is different than the additive phenotype of the double mutants described by Hattori et al. (1992) and Mourad et al. (1995). It is unclear whether the high level of SU- R expressed by the S4-Hra double mutant was the consequence of the naturally high level 187 of resistance characteristic of the second mutation (tryptophan to leucine) or a synergistic interaction of the proline to alanine plus tryptophan to leucine mutations. Ott et al. (1996) have recently described a molecular model for the ALS enzyme based on its homology and suggested ancestral relationship with pyruvate oxidase. A model of the Arabidopsis ALS homodirner was derived from the X-ray crystal structure of the Lactobacillus planatarum pyruvate oxidase protein (Muller and Schulz 1993 ), primary and computer-predicted secondary ALS structure, and structure-activity relationship data available fi'om IMI analogue screens. The utility of the model was demonstrated by the rational design of three novel ALS mutations resulting in IMI-R enzyme activity (Ott et al. 1996; Kakefuda et al. 1996). Interestingly, the functional equivalent amino acid residues to the sugarbeet sites alanine 113 and proline 188 were predicted to be in close proximity to one another. Kakefuda et al. (1996) further propose both of these residues are required to correctly position a strictly conserved lysine residue within the herbicide binding pocket. The positively charged lysine residue is predicted to interact with the carboxyl group of IMI herbicides and anchor the herbicide within the binding pocket (Kakefuda et al. 1996). The hydr0phobic nature of the alanine and proline residues apparently interact with the lysine side chain to correctly position the positively charged terminal amino group. By altering the alanine or proline residues to more polar residues threonine and serine, respectively, individual mutations may Slightly alter the lysine position and therefore herbicide binding and sensitivity. Additionally, the double mutant, 93R3OB, would incorporate the substitution of two polar residues for alanine and proline. The synergistic effect of the two mutations in 93R3OB is consistent with the proposed model and attributed filnctions of the specific amino acid residues. The serendipitous selection of 188 two single mutations and the cognate double mutant has served to validate a usefiil model of the ALS herbicide binding pocket. With the full compliment of alleles (wt, two class-specific single resistance mutations, and one cross resistant double mutation) in Figure 6, an excellent opportunity exists to examine the interaction of mutations within the herbicide binding domain of ALS. Also, the selection fiom tissue culture of these resistance traits avoids many of the regulatory hurdles faced bringing transgenic herbicide resistant crops into production. Finally, these somaclonal selections may provide an excellent tool for sugarbeet producers to avoid ALS-inhibiting herbicide carryover injury to sugarbeets, shorten sugarbeet crop rotation restrictions, increase flexibility in cropping rotations, and provide the opportunity to use proven herbicides as new weed control tools in sugarbeets. ACKNOWLEDGEMENTS The authors would like to thank Steve Stumer and Dr. Newell Bascomb for advice and assistance in automated and manual gene sequencing studies. This research was financially supported by American Cyanamid. 189 LITERATURE CITED . Anderson, P. C. and M. Georgeson. 1989. Herbicide-tolerant mutants of corn. Genome 31:994-999. . Anonymous. 1991. Preparation of genomic DNA fiom plant tissue. Pages 2.3.1-2.3.3 in F.M. Ausubel, R. Brent, R E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds. Current Protocols in Molecular Biology. John Wiley and Sons Publishing, New York. . Bedbrook, JR, R. S. Chaleff, S. C. Falco, B. J. 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Woodworth, A., P Bemasconi, M. Subramanian, and B. Rosen. 1996b. A second naturally occurring point mutation confers broad-based tolerance to acetolactate synthase inhibitors. Plant Physiol. 11128105. 66. Wright, T. R. and D. Penner. 1996. Mechanism of imidazolinone resistance of two sugarbeet somaclonal selections. Proc. North Cent. Weed Sci. Soc. 51:98. 67 . Wright, T. R. and D. Penner. 1997. Selection and genetic analysis of two imidazolinone-resistant sugarbeets. Abstr. Weed Sci. Soc. Amer. 37:253. 198 68. Yadev, N., R E. McDevitt, S. Benard, and S. C. Falco. 1986. Single amino acid substitutions in the enzyme acetolactate synthase confer resistance to the herbicide sulfometuron methyl.» Proc. Natl. Acad. Sci, USA 83:4418-4422. 199 60:000. .0: 003 00303000 2&0? 50¢ 82?. {cow 00:3 023% 000—0 000ng 05 :05 08003 3 003— mm 02.5 e .3330.“ 00: 3 002050: 000303 0859200 3030380 < Amodudv hugged 0% 00: 0.0 80:80:02.8 3 000065 See 38 £536 0:0. 089. 05 .3 330:8 Sea a .3 95 0.525 74mm 033800 05 8 550—0.... E03000 £000 «0 3 we owe.— 0053000 n mg a 29:80 020300: 0: 0:00? {can $2000 030 003000 8 0003—000 5:823:00 020300: 05. . e mnfle e $32 a 888 a 838 9285 858:8 $3 $2000 088% mi? .qAfiouoa was TE 3300—3000 BE: 0030.833 0850 0055a 335; Mo 53:00 0500mm. X com 0 on X em a m X m 0 Rd .1 0 S .o m... 80330835 X co? 0 cm X 02: a A. X m a 25.0 r: eSod Dm Shaina—:0 X 82A 0 eooomA X _ a m X ov a com .1 on m a): cat—0530:: 93 A23 93 93 m2 3 m3 3 ma 3 Lmb— .3 3.0—0 0203003 0028 am new 05 3mm 003325 «0 000020 00.50 3 Seen—«00 0850 mi? .~ 0305 200 Figure I. Deduced amino acid substitutions within the ALS enzyme primary structure of selected plant species resulting in resistance to one or more classes of ALS-inhibiting herbicides. One letter amino acid codes within the box represent a change from the highly conserved amino acids listed in the model ALS enzyme above. The N—terminal chloroplast transit peptide (CTP) represents an approximately 85 amino acid portion (Bedbrook et al. 1995; Bemasconi et al. 1995; Grula et al. 1995) of the enzyme required to direct nuclear-coded proteins to the chloroplast. The CTP is cleaved upon import into the chloroplast to form the mature ALS enzyme. Arrows indicate PCR primers used to amplify fragments corresponding to Region A and Region B from sugarbeet genomic DNA. 3' 3* “*Hw’f—l 201 ................................................. Xanthium stmmarium L.” Zea mays L.° Arabidopsis thaliana (L.) Heyhn " S Lactuca serriola L.° H Nicotiana tabacum L.f Q Xantr'um stmmarium L.‘ V Amaranthus .sp. " Nicotiana tabacum L.f A Xantium strumarium L.” Zea mays L.” Arabidopsis thaliana (L) Heyhn.i Zea mays L.°" El . "-"Sl . l . .l .l. l 315 } T T l"t""l"‘l" 22 'CTP, chloroplast transit peptide. ”Bemasconi et al. (1995). °Bright et al. (1992). “Haughn et al. (1988). °Guttieri et al. (1992). fLee et al. (1988). ”Woodworth et al. (1996a). Woodworth et al. (1996b). fSathsavian et al. (1991). JDietrich (1992). 202 Figure 2. \Vrldtype (wt) sugarbeet ALS nucleotide and deduced amino acid sequences (nucleotide positions 301-660; amino acid positions 101-220) surrounding and including Region A (shaded) fi'om Figure 1. Coding strand is line A; noncoding strand, line B; and deduced amino acid sequence, line C. The three herbicide resistance residues listed in Figure 1 for Region A are indicated by a box around the deduced amino acid. Arrows indicate the PCR primers, ForA and RevA, used to specifically amplify the Region A gene fragment fi'om genomic DNA for sequencing. 203 ...................................................................................... ..................................................................................................... s cnccmuncmmrcm‘mc'rrnccc 301 t - * iii; ZZZ 360 3 can “A” can no 101scvrnvrnypcclsnsraqn 120 121 L T R S K T I R N V L P R H E Q G G V P 140 141 A A E G Y A R A T G K V G V C I A T S G 160 481 .. ,, V. _. .. ,. 161 5 4 1 :1: is: it: 3:51: -j;:E:.S:z;};~;:;~;::x:-l ::£::3f,2:;,.vf:. 181VAITGQV|§IRRMIGTDEIFQET zoo CCAATTGTTGAGGTGACAAGGTCTATTACTAAGCATAATTATTTAGTTTTGGATGTAGAG 3' 601 + + + + ¢ ¢ 660 qufAACAACTCCACTGTTCCAGATAATGATTCGTATTAATAAATCAAAACCTACATCTC 5' RevA 201 P I V E V T R s I T K H N Y L V L D V E 220 204 Figure 3. Wildtype (wt) sugarbeet ALS nucleotide and deduced amino acid sequences (nucleotide positions 1621-2027; amino acid positions 541-665) surrounding and including Region B (shaded) from Figure 1 (note stop codon at nucleotide 1996-8 indicated by * in line C). Coding strand is line A; noncoding strand, line B; and deduced amino acid sequence, line C. The two herbicide resistance residues listed in Figure 1 for Region B are indicated by a box around the deduced amino acid. Arrows indicate the PCR primers, ForB and RevB, used to specifically amplify the Region B gene fragment from genomic DNA for sequencing. _ ”’7‘ 205 3028 A. 1680 B. C. 560 580 if 1800 C. 581 T Y L G N P S K S A D I F P D M L X F A 600 ' “ 1. H,. ..mmwwmfi 1860 mmwhmcwmmccmeacccacacnmmaact??? it: 39:17:: 1; 1:; ii 31* C. 601 E A C D I P S A R V S N V A D L R A A I 620 7? 1920 C. 621 Q T M L D T P G P Y L L D V I V P H Q E 640 A. 1921 B. ,mmfi: 1980 c. 641 a v L P u I P [3 c a c r K 0 T I T e 0 0 - A. GGAAGAACCTCTTATTGATCGGTTTAATGACGGTTGGAACCATTTAA 3' 1981 + + — —+ + — 2227 3. cc TCTTGGAGAATAACTAGCCAAATTACTGCCAACCTTGGTAAATT 5' RevB C. 661 G R T S Y * 665 206 '.\ Figure 4. Metabolism of parent [“C]-imazethapyr and accumulation of the a- hydroxyethyl-imazethapyr metabolite in four sugarbeet lines. No significant difference in rate of imazethapyr metabolism was detected (0t=0.05). Radioactivity extracted (%) 207 100 Parent imazethapyr +19! 80 —l— Sur + Sir-13 60 + 93R30B OH-imazethapyr 40 a; ,.:=;-,6 ' ' '9 ' ' Wt .aak’é‘z‘." ...... Sur 20 12.-’3“ -----A~ Sir-13 O léiiiiiiiiiinnu .auu::2!‘.‘.':':':':':':’. ' ° '5' " 93R303 O 5 24 95 Time after treatment (h) 208 Figure 5. Sequencing gel of sugarbeet ALS Region A showing two nucleotides where resistant lines showed one or more changes from the wildtype (wt) sequence. 209 G3,37 IfiAm in nucleotide sequence yields A113 ‘I‘m in deduced amino acid sequence , Plsg’Sm in deduced . amino acid sequence 93R30B 210 Figure 6. Deduced amino acid composition for three ALS-resistant sugarbeet selections and a sensitive wildtype (wt) sugarbeet at the five known plant ALS sites for herbicide resistance. Amino acids different from the wildtype residue are identified with a box. Corresponding ALS-inhibitor class resistance is listed for each sugarbeet selection. 211 Herbicideclass .Slr-"Sl'l' 1.1.1515 ----IMISUTP wt A P A W S S S S Sir-13 P A w s R s s Sur A [3] A w s s R R 7'" 93R3OB [E A w s R R R Chapter 5 SUMMARY STATEMENTS 212 213 Positive impacts of imidazolinone-resistant sugarbeets. The development of three sugarbeet selections with varying cross resistance phenotypes may have many potentially positive consequences for sugarbeet producers. First, the collection of resistance alleles Sur (with the P133 to S mutation), Sir-13 (A113 to T), and 93R3OB (A113 to T plus Pm to S) represent the first time class-specific ALS-inhibiting herbicide resistance mutations have been selected independently and subsequently isolated in double mutant form resulting in broad cross resistance. Additionally, the 93R308 double mutant expresses a higher level of resistance at the enzyme level than the sum of the single mutants to specific herbicides from the SU, IMI, and TP herbicide classes. The synergistic interaction of these two mutation sites may provide an excellent opportunity to extensively examine the specific interactions of multiple ALS-inhibiting chemicals with the enzyme. As discussed in the results and discussion section in chapter 4, the phenotypes of the various sugarbeet selections are consistent with similar mutations in other species. Also, the interaction of the two mutation sites agrees with the predicted location of these amino acid residues within the herbicide-binding site of a computer-generated ALS-herbicide binding site (Ott et al. 1996; Kakefuda 1996). Additional detailed examination of the interaction of each ALS variant enzyme form with a series of ALS-inhibiting chemicals may provide further insight into the interplay of these mutation sites. On a more applied scale, these herbicide resistant selections may successfully address the agronomic problems which were the goal of this project. In greenhouse experiments, IMI-R in Sir-13 showed sufficient magnitude of resistance in surviving greater than field rate equivalent soil residues of imazethapyr and imazamox. In an unreplicated preliminary greenhouse experiment, both Sir-13 and 93R3OB showed 2 14 suflicient resistance to survive soil residues of field rate equivalent doses of imazethapyr, imazamox, imazapyr, and imazaquin (data not shown). The implications are that the lengthy rotation restrictions for sugarbeets with M herbicides may be shortened or eliminated with Sir-13 and 93R3OB. Additionally, 93R3OB showed cross resistance to soil residues of several SU and TP herbicides which can reduce the threat of carryover injury to sugarbeets from these classes of ALS-inhibiting chemistries as well (data not shown). The reduction or elimination of rotation restrictions for ALS-inhibiting herbicides with IMI-R/SU-R sugarbeets may increase the crop rotation flexibility for sugarbeet producers. This may shorten the crop rotation strategy so that sugarbeets (a high value crop) can be produced more fi'equently, may increase the crops that can be grown in rotation, or allow flexibility in the order of crops grown in rotation. The impact of overcoming the ALS-inhibitor carryover problem is significant and was the primary goal of the project; however, herbicide resistance with 93R3OB, especially, appears high enough to consider the use of specific ALS-inhibiting herbicides as weed control options in sugarbeets. This could positively affect sugarbeet production in a variety of ways. Broad spectrum weed control might be achieved by a single POST spring herbicide application (not necessarily a single herbicide) rather than multiple reduced-rate applications plus field cultivations. The persistence of ALS-inhibiting herbicides provides extended weed control of late-germinating weeds. The impact will be to reduce the time, labor, and amount of chemical utilized for weed control in sugarbeets. Other biotechnologically-improved sugarbeets will be marketed in the near fiiture, including glufosinate- and glyphosate-resistant hybrids (Maruska et al. 1996; Hart et al. 1996). These herbicide resistance traits are genetically engineered and although the mode 215 of action of each of these herbicides is distinctly different, their effect in crop production will be very similar. Each herbicide is considered a non-selective herbicide except in the case of engineered crop resistance. But neither herbicide possesses soil residual activity. Therefore, while these herbicides may provide excellent control of emerged weeds at the time of application (which must be applied POST), multiple applications will be required to control late-germinating weeds. For this reason, resistance to soil-active herbicides, like the IMI herbicides, is advantageous versus resistance to glufosinate and glyphosate sugarbeets. Additionally, the glufosinate- and glyphosate-resistant sugarbeets are genetically engineered, a trait which currently is unfavorable in many world markets, especially in the European Union states where genetically modified organisms (GMO) are considered tainted or unsafe. In this vein, the IMI-R and SU-R sugarbeets, which were derived by non-transgenic means, would be more accepted worldwide. This point should be considered in the context that 75% of the world sugarbeet production occurs in Europe. In the case of all three types of herbicide resistant sugarbeets, a very important attribute may be the increased crop tolerance to these herbicides compared to herbicides currently used in sugarbeet production. The potential exists to avoid sugarbeet injury and yield losses caused by current chemical weed control practices as observed by Dexter (1994) and Starke and Renner (1996). By creating a high margin of crop safety to herbicides that control the same or broader weed spectrum, sugarbeet yields may be increased. One potentially positive aspect of ALS-inhibitor resistance that was not addressed in this work is the prospect of a pleiotropic effect of more rapid seed germination associated with ALS-inhibitor resistant biotypes. Researchers have observed SU-R 216 Kochia and Lactuca spp. germinated at a faster rate than susceptible counterparts, especially at lower temperatures (Alcocer-Ruthling 1992; Dyer et al. 1993). Additionally, cumulative germination of these wild species was 99 to 100%. Ifthese traits were associated with the herbicide resistance trait in sugarbeet, production could be impacted in a very positive manner by the IMI-R and SU-R sugarbeets. Early germination at lower temperatures has been a goal that sugarbeet breeders have had little success in obtaining (Bosemark 1993). Additionally, better seed germination is a must for uniform plant stand establishment which aids in weed control by utilizing what competition sugarbeets can ofl‘er against weeds that are present. Higher yields are correlated with longer growing seasons (Bosemark 1993 ), implying that more rapid establishment of sugarbeets planted earlier (during cooler periods) may be able to increase sugarbeet yields. Negative impacts of imidazolinone-resistant sugarbeets. Of the potential negative attributes of ALS-inhibiting herbicide resistant sugarbeets, one may be reduced productivity of the resistant plants. Weed resistance to triazine herbicides clearly has detrimental physiological consequences due to reduced efficiency of photosystem II in the light reactions of photosynthesis (Conard and Radosevich 1979; Ort et al. 1983). In general, whole plant fitness differences between ALS-inhibitor resistant and sensitive biotypes have been difficult to demonstrate unequivocally. It is expected that no fitness or yield penalty would be associated with herbicide resistance trait, as seen with other ALS- inhibitor resistant crops; however, in chapter 4, a lower ALS specific activity was reported for enzyme extracts from each of the herbicide-resistant selections. Again, these results are somewhat in doubt since the plants examined were non-isogenic. Future studies to 21 7 examine this phenomenon should be conducted once the herbicide resistance traits have been back-crossed several generations into elite sugarbeet lines. Another potential impact of these sugarbeets could be the rapid selection of ALS- based herbicide resistance in weeds if ALS-inhibiting herbicides are used in sugarbeets in addition to rotational crops for weed control. This most be addressed in terms of production management, incorporating a sound herbicide resistance prevention/management strategy to avoid fiirther proliferation of herbicide resistant weeds. Another potential mechanism to create herbicide-resistant weeds is by direct outcrossing to weedy relatives. In the United States, no known relatives freely cross pollinate with sugarbeets; however, in Europe, weedy wild beets (Beta maritima L., Beta macrocaIpa L., and Beta atriplicafolia Rouy) are currently present in sugarbeet production fields and will interpollinate with sugarbeets (Smith 1980; Boudry et al. 1993; Bosemark 1993). Good management practices utilizing other weed control measures, especially in seed production fields should prevent transmission to of the resistance trait to weed beets. In fact, Bosemark (1993) suggests this may allow greater control of weed beets within sugarbeet fields. Since sugarbeet is a biennial crop harvested the first year, in the vegetative state, cross pollination should not be a consideration except in seed production fields. The potential to shorten crop rotations could impact sugarbeet yields negatively if pest pressures (disease, insects, and nematodes) become too great by reducing crop diversity in the rotation. To a great extent, these pests are currently managed by the cultural practice of crop rotation. 21 8 LITERATURE CITED . Alcocer-Rutherling, M., D. C. Thill, and B Shafii. 1992. Seed biology of sulfiionylurea-resistant and -susceptible biotypes of prickly lettuce (Lactuca serriola). Weed Technol. 62858-864. . Bosemark, N. O. 1993. Genetics and breeding. Pages 66-119 in D. A Cooke and R. K. Scott eds. The Sugar Beet Crop. Chapman and Hall, London, England. . Boudry, P. M. Morchen, P. Saumitou-Laprade, P. Vemet, and H. VanDijk. 1993. The origin and evolution of weed beets: consequences for the breeding and release of herbicide-resistant transgenic sugar beets. Theor. Appl. Genet. 87:471-478. . Conard, S. G. and S. R. Radosevich. 1979. Ecological fitness of Senecio vulgaris and Amaranthus retroflexus biotypes susceptible and resistant to atrazine. J. Appl. Ecol. 16: 17 1-177. . Dexter, A. G. 1994. History of sugarbeet (Beta vulgaris) herbicide rate reduction in North Dakota and Minnesota. Weed Technol. 8:334-337. . Dyer, W. E., P. W. Chee, and P. K. Pay. 1993. Rapid germination of sulfonylurea- resistant Kochia scoparia L. accessions is associated with elevated seed levels of branched chain amino acids. Weed Sci. 41:18-22. . Hart, S. E. 1996. Risks and challenges associated with the use of herbicide resistant/tolerant crops. Proc. North Cent. Weed Sci. Soc. 51:175-176. . Kakefuda, G., K.-H. Ott, J.-G. Kwagh, and G. W. Stockton. 1996. Structure-based designed herbicide resistant products. World Pat. Appl. No. WO96/33270. 219 9. Maruska, K., J. Staska, . B. Thomess, and P. G. Mayland. 1996. Glufosinate for weed control in transgenic sugarbeet. Proc. North Cent. Weed Sci. Soc. 51:149. 10. Ort, D. R., W. H. Ahrens, B. Martin, and E. W. Stoller. 1983. Comparison of photosynthetic performance in triazine-resistant and susceptible biotypes of Amaranthus hybridus. Plant Physiol. 72:925-930. 11. Smith, G. A 1980. Sugarbeet. Pages 601-616 in W. R Fehr and H. H. Hadley, eds. Hybridization of Field Crops. Amer. Agron. 800. and Crop Sci. Soc. Amer., Madison, WI. 12. Ott, K.-H., J .-G. Kwagh, G. W. Stockton, V. Sidirov, and G. Kakefiida. 1996. Rational molecular design and genetic engineering of herbicide resistant crops by structure modeling and site-directed mutagenesis of acetohydroxyacid synthase. J. Mol. Biol. 263:359-368. 13. Starke, R. J. and K. A. Renner. 1996. Velvetleaf (Abutilon theophrasti) and sugarbeet (Beta vulgaris) response to triflusulfiiron and desmedipham plus phenmedipham. Weed Technol. 10:121-126. APPENDIX A ALS-INHIBITING HERBICIDE CHEMICAL NAMES 220 221 Chemical names for currently marketed and experimental ALS-inhibiting herbicides. Common name Chemical name Amidosulfuron N-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]- N-methylmethanesulfonamide Azimsulfuron N-[[(4,6-dimethoxy-s-pyrimidinyl)amino)carbonyl]-l-methyl-4-(2- methyl-ZH-tetrazol-S-yl}lH-pyrazole-S-sulfonamide Bensulfuron 2-[[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino] sulfonyl]methyl]benzoic acid Chlorimuron 2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]amino] sulfonyl]benzoic acid Chlorsulfiiron 2-chloro-N-[[(4-methoxy-6-methyl-1 ,3 , 5-triazin-2-yl)amino]carbonyl] benzensulfonamide Cinosulfiiron N-[[(4,6-dimethoxy-l,3,5-triazin-2-yl)amino]carbonyl]-2-(2- methoxyethoxy)benzenesulfonamide Cloransulam N-(2-carboxymethyl-6-chlorophenyl)-5-ethoxy-7-fiuoro[1,2,4] trizolo[ 1 , 5c]pyrimidine-2-sulfonamide Cyclosulfamuron N-[[[2-(cyclopropylcarbonyl)phenyl]amino]sulfonyl]-N ’-(4,6- dimethoxy-Z-pyrimidinyl)urea Ethametsulfuron 2-[[[[[4-ethoxy-6-(methylamino)-1,3,5-triazin-2-yl]amino]carbonyl] amino]sulfonyl]benzoic acid Flazasulfiiron N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3- (trifluoromethyl)-2-pyridinesulfonamide 222 Common name Chemical name Flumetsulam Halosulfiiron Imazameth Imazamethabenz Imazarnox Imazapyr Imazaquin Imazethapyr Imazosulfirron KIH-2023 N-(2,6-difluoroethyl)-5-methyl[l,2,4]triazolo[l,5a]pyrimidine-2- sulfonamide Methyl 5-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl] aminosulfonyl]-3 -chloro- 1 -methyl- 1 -H-pyrazole-4-ca.rboxylate (¢)-2-[4,5-dihydro-4-methyl-4-(l-methylethyl)-5-oxo-lH-imidazol-Z- yl]-5-methyl-3-pyridinecarboxylic acid (:t)-2-[4,5-dihydro-4-methyl-4-(l-methylethyl)-5-oxo-1H-imidazol-2- yl]-4(andS)-methylbenzoic acid (3 :2) 2-(4-isopropyl-4-methyl-S-oxo-2-irnidazolin-2-yl)-S-(methoxymethyl) nicotinic acid (i)-2-[4, S-dihydro-4-methyl-4-( 1-methylethyl)-5-oxo- lH-imidazol-Z- yl]-3 -pyridinecarboxylic acid 2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-lH—imidazol-Z-yl]- 3-quinolinecarboxylic acid 2-[4, 5-dihydro-4-methyl-4-(1 -methylethyl)-5 -oxo- lH-imidazol-2-yl]- 5-ethyl-3 -pyridinecarboxylic acid 1-(2-chloroimidazol[l,2-a]pyridin-3-sulfonyl)-3-(4,6- dimethoxypyrimidin-Z-yl)urea Sodium 2,6-bis[(4,6-dimethoxypyrimidin-Z-yl)oxy]benzoate 223 Common name Chemical name KIH-6127 Metosulam Metsulfirron Nicosulfuron Oxasulfuron Primisulfuron Prosulfiiron Pyrazosulfuron Pyrithiobac Rimsulfuron 8-chloro-7-fluoro-1 1, 12, 13,13a-tetrahydro-9-oxo-9H-imidazol [l,5-a]pyrrolo[2, 1-c][1,4]benzodiazepine-carboxylic acid N-(2,6-dichloro-3 -methylphenyl)-5,7-dimethoxy[1,2,4]triazolo[1,5a] pyrimidin-Z-sulfonamide 2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino] sulfonyl]benzoic acid 2-[[[[(4,6-dimethoxy—2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]- MN-dimethyl-3-pyridinecarb0xamide 2-[[[[(4,6-dimethyl-2-pyrimidinyl)amin0]carbonyl]amino]sulfonyl] benzoic acid 2-[[[[[4,6-bis(difluoromethoxy)-2-pyrimidinyl]amino]carbonyl] amino]sulfonyl]benzoic acid 1-(4-methoxy-6-methyl-triazin-2-yl)-3-[2-(3,3,3-trifluoropropyl) phenyl-sulfonyl]-urea 5-[3 -(4,6-dimethoxypyrimidin-2-yl)-carbonylsulfamoyl]- l - methylpyrazole-4-carboxylic acid 2-chloro-6-[(4,6-dimethoxy—2-pyrmidinyl)thio]benzoic acid N—[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)- 2-pyridinesulfonamide 224 Common name Chemical name Sulfometuron 2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl] benzoic acid Sulfosulfiiron 1-(2-ethylsulfonylimidazol[ l ,2-a]pyridin-3 -ylsulfonyl)-3 -(4,6- dimethoxypyrimidin-Z-yl) urea Thifensulfiiron 3 -[[[[(4-methoxy-6-methyl-l ,3 , S-triazin-2-yl)amino]carbonyl]amino] sulfonyl]-2-thiophenecarboxylic acid Triasulfuron 2-(2-choroethoxy)-N-[[(4-methoxy-6-methyl-1,3,5-tn'azin-2-yl) amino]carbonyl]amino]benzenesulfonamide Tribenuron 2-[[[[(4-methoxy-6-methyl- 1,3 , 5-triazin-2-yl)amino]carbonyl]amino] sulfonyl]benzoic acid Triflusulfuron 2-[[[[[4-(dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl] amino]carbonyl]amino]sulfonyl]-3-methylbenzoic acid APPENDIX B ONE- AND THREE-LETTER ABBREVIATIONS FOR AMINO ACIDS 225 226 One- and three-letter abbreviations for amino acids. One Three letter letter Amino acid A Ala Alanine C Cys Cysteine D Asp Aspartate E Glu Glutamate F Phe Phenylalanine G Gly Glycine H Hrs Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine 227 One- and three-letter abbreviations for amino acids One Three letter letter Amino acid V Val Valine W Trp Tryptophan Y Tyr Tyrosine