Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 35-40). Also available on the World Wide Web.

Studies were conducted in 1996 through 1999 to: (1) evaluate the responses of one imidazolinone (IMI)-susceptible (S) and four -resistant (R1, R2, R3, and R4) smooth pigweed populations to various acetolactate synthase (ALS)-inhibiting herbicides, (2) determine the mechanism of resistance, and (3) evaluate the relative growth and competitiveness of each population. Field studies were conducted in 1996 near Marion, MD, in a field with a history of repeated imazaquin use. Smooth pigweed control with IMI herbicides was < 8 percent, but control with sulfonylurea (SU) herbicides ranged from 73 to 99 percent. Follow-up greenhouse studies were used to confirm IMI resistance in the Marion, MD smooth pigweed population (R4) as well as three others (R1, R2, and R3). R populations were 730- to 1350-fold more tolerant to imazethapyr than the S population. Based on resistance ratios, all R populations displayed low-level cross-resistance to chlorimuron and negative cross-resistance to thifensulfuron, pyrithiobac, and cloransulam-methyl with R2 being the most sensitive of the R populations to pyrithiobac and cloransulam-methyl. Absorption, translocation, and metabolism of ¹⁴C-cloransulam-methyl in S and R2 populations were generally similar. Three metabolites of cloransulam-methyl with ratio of front (Rf) values approximately 0.83, 0.65, and 0.45 were isolated. The metabolite with a 0.83 Rf value increased over time as the parent molecule decreased indicating that it plays a major role in cloransulam-methyl metabolism in smooth pigweed. The other metabolites did not change significantly over time and never represented more than 5 percent of the extracted radioactivity. The identity of these metabolites has not been determined. Using enzyme assays, it was determined that IMI resistance in R populations was due to an altered ALS that was no longer susceptible to inhibition by these herbicides. ALS from S, R1, and R2 populations responded similarly to chlorimuron and thifensulfuron, but reductions in enzyme activity by chlorimuron and thifensulfuron were significantly greater for R3 ALS than for S, R1 or R2 ALS. ALS from R2 and R3 was significantly more sensitive to inhibition by pyrithiobac compared to S ALS. Based on resistance ratios, R2 and R3 ALS were also more sensitive to inhibition by cloransulam-methyl than S ALS. Negative cross-resistance to thifensulfuron, pyrithiobac, and cloransulam-methyl in some R populations at the whole-plant level can be explained by increased sensitivity at the enzyme level. Under noncompetitive conditions in the greenhouse, S produced 17, 23, 25, and 44 percent more biomass than R1, R2, R3, and R4 populations, respectively. S plants were also taller than R plants 17 and 21 d after planting (DAP) and displayed a faster initial rate of leaf area increase compared to all R populations. The net assimilation rate of S was significantly higher than R2 and R3 populations 24 DAP. R3 and R4 populations had significantly less chlorophyll per g of plant tissue compared to S; therefore, reduced growth in some R populations compared to S may be linked to chlorosis that generally appears early in seedling development. Biomass production in the field under competitive conditions was similar for all populations using both monoculture and mixed populations. For this reason, the differences in growth observed in the greenhouse in the S population may not confer a competitive advantage over R populations in the field. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / Acetolactate synthase (ALS) and acetohydroxyacid synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that catalyze the formation of acetolactate from two molecules of pyruvate. In addition to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important precursors to the essential amino acids valine and isoleucine. Conversely, ALS forms acetolactate as a precursor to 2,3-butanediol, a product formed in an alternative pathway to mixed acid fermentation. While these enzymes catalyze the same reaction, they have been found to be quite different. Such differences include: biological function, pH optimum, cofactor requirements, reaction kinetics and quaternary structure. Importantly, AHAS has been identified as the target of the widely-used sulfonylurea and imidazolinone herbicides, which has led to many structural and kinetic studies on AHAS enzymes from plants, bacteria, and fungi. ALS, on the other hand, has only been identified in bacteria, and has largely not seen such extensive characterization. Finally, although some bacteria contain both enzymes, they have never been studied in detail from the same organism. Here, the ALS and AHAS enzymes from Klebsiella pneumoniae were studied using steady-state kinetic analyses, X-ray crystallography, site-directed and site-saturation mutagenesis, and cell growth complementation assays to i) compare the kinetic parameters of each enzyme, ii) compare the active sites to probe their differences in substrate profile and iii) test the ability of ALS to function in place of AHAS in vivo.

Acetolactate synthase (ALS) inhibiting herbicides are commonly used to eliminate weeds from mature turfgrasses. Field trials were conducted from 2004-2006, testing ALS herbicides for preemergence and early postemergence activity on newly seeded turfgrasses, using four species: Riviera bermuda, Zenith and Companion zoysia, L- 93 creeping bentgrass, and Poa annua L. Data collected were phytotoxicity and percent turf cover. Bermuda and zoysia herbicides were metsulfuron-methyl (42 g ha-1), trifloxysulfuron (29 g ha-1), flazasulfuron (53 g ha-1), foramsulfuron (30 g ha-1), bispyribac-sodium (112 g ha-1), and rimsulfuron (35 g ha-1). Treatments occurred the day of seeding and two-three weeks after seeding. Flazasulfuron, trifloxysulfuron and bispyribac-sodium caused significant damage in all treatments. Data suggests that bermuda and zoysia are tolerant of seedling treatments of foramsulfuron, rimsulfuron, and metsulfuron-methyl at these rates. Bentgrass and P. annua herbicides were foramsulfuron (15 and 30 g ha-1), siduron (2803 g ha-1), bispyribac-sodium (49 g ha-1), and paclobutrazol (281 g ha-1). Treatments occurred the day of seeding, two and four weeks after seeding. Foramsulfuron at 15 and 30 g ha-1 caused significant damage regardless of when it was applied. Data suggests that bentgrass and P. annua are tolerant of seedling treatments of siduron, paclobutrazol, and bispyribac-sodium at these rates.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12881号 / 論農博第2808号 / 新制||農||1028(附属図書館) / 学位論文||H26||N4880(農学部図書室) / 31599 / (主査)教授 冨永 達, 教授 奥本 裕, 教授 宮川 恒 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p>Acetolactate synthase (ALS) and acetohydroxyacid synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that catalyze the formation of acetolactate from two molecules of pyruvate. In addition to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important precursors to the essential amino acids valine and isoleucine. Conversely, ALS forms acetolactate as a precursor to 2,3‑butanediol, a product formed in an alternative pathway to mixed acid fermentation.</p> <p>While these enzymes catalyze the same reaction, they have been found to be quite different. Such differences include: biological function, pH optimum, cofactor requirements, reaction kinetics and quaternary structure. Importantly, AHAS has been identified as the target of the widely-used sulfonylurea and imidazolinone herbicides, which has led to many structural and kinetic studies on AHAS enzymes from plants, bacteria, and fungi. ALS, on the other hand, has only been identified in bacteria, and has largely not seen such extensive characterization. Finally, although some bacteria contain both enzymes, they have never been studied in detail from the same organism. </p> <p>Here, the ALS and AHAS enzymes from <i>Klebsiella pneumoniae</i> were studied using steady-state kinetic analyses, X-ray crystallography, site-directed and site‑saturation mutagenesis, and cell growth complementation assays to i) compare the kinetic parameters of each enzyme, ii) compare the active sites to probe their differences in substrate profile and iii) test the ability of ALS to function in place of AHAS <i>in vivo</i>.</p>

Graduation date: 2004

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 96-107).

Doctor of Philosophy / Department of Agronomy / Tesfaye T. Tesso / The lack of effective post-emergence weed control options is often highlighted as one of the major factors behind dwindling acreage under sorghum (Sorghum bicolor (L.) Moench) in the United States. The discovery of herbicide resistance sources in wild sorghum population and subsequent efforts to incorporate them into cultivated sorghum was received with much optimism to change weed management practices in sorghum. As the development of the technology advances, especially of the Acetolactate synthase (ALS) resistance, concerns over the temporary interveinal chlorosis and reduced seedling vigor in some of the resistant families became heightened. This thesis research is designed to shed light on the genetic basis of seedling chlorosis and assess its impacts on yield potential. The study has three parts; the first part is focused on identifying the genetic causes and plant mechanisms associated with the chlorotic phenotype. ALS herbicide resistant sister-lines expressing normal and chlorotic phenotypes were analyzed via RNA sequencing at four time points during seedling growth. The study identified several variants of genes coding chloroplast precursors and those that cause epigenetic modifications. Once confirmed, genetic markers can be developed to track these gene variants in the breeding population and eliminate segregates genetically prone to chlorosis/yellowing. The second part of the study focuses on assessing the effect of ALS resistance associated chlorosis on agronomic and nutritional parameters of sorghum inbred lines. A set of ALS resistant lines expressing different levels of the chlorotic phenotype were evaluated in replicated field trials and laboratory methods. Results showed that interveinal chlorosis delays flowering but does not have negative effect on yield and nutritional parameters with and without herbicide treatment. The last part addresses whether there is any yield drag that may be associated with herbicide resistance traits and foliar interveinal chlorosis. For this, we synthesized a large set (182) of hybrids from ALS resistant, ACCase resistant and regular (susceptible) seed and pollinator parents. The hybrids were then evaluated in three sets at multiple locations during the 2014 and 2015 crop seasons along with commercial checks. The results revealed that resistance to both herbicides do not cause any drag to grain yield. The traits also do not have any negative impact on grain and nutritional quality of resistant hybrids.

<p>  </p> <p>Giant ragweed (<em>Ambrosia trifida</em> L.) is an annual broadleaf plant capable of emergence throughout the cropping season, opportune colonization of disturbed soil, rapid biomass accumulation, and a propensity to evolve mutations that endow resistance to herbicides, all of which contribute to giant ragweed being one of the most challenging weeds to control in row-crop production. Many soybean growers rely on acetolactate synthase (ALS)-inhibiting herbicides such as cloransulam for control of giant ragweed prior to its emergence, though the spread of biotypes resistant to ALS inhibitors can render these herbicides largely ineffective. Mesotrione inhibits the 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme, and applications of this herbicide have recently been approved for use in mesotrione-resistant soybean varieties. Field experiments demonstrated that preemergence applications of mesotrione resulted in greater control of giant ragweed populations segregating for ALS-inhibitor resistance than several other commonly used herbicide combinations. Where mesotrione was applied, giant ragweed biomass was reduced by an average of 84% relative to the nontreated, while treatments without mesotrione increased biomass by an average of 34% by suppressing competition from other weed species. Additionally, both soil- and agar-based bioassays demonstrated that combinations of mesotrione and metribuzin can be synergistic for control of giant ragweed. </p> <p>Cloransulam was shown to result in strong selection for giant ragweed individuals with ALS-inhibitor resistance, increasing the proportion of resistant plants that emerged at one field site from 15% to greater than 90% after a single preemergence application. This selection pressure was reduced when mesotrione was co-applied with cloransulam. However, no herbicide combination, including sequential applications of non-ALS-inhibiting herbicides, consistently resulted in a resistance frequency similar to the baseline if an ALS inhibitor was applied preemergence. Resistance to cloransulam and other ALS inhibitors is expressed in giant ragweed plants possessing at least one mutant (Trp574Leu) <em>ALS</em> allele. The distribution of this allele in one field violated the Hardy-Weinberg Equilibrium, despite the fact that <em>ALS</em> is a nuclear gene and the Trp574Leu mutation does not incur a fitness penalty. We suspected that the inheritance of this mutation may be linked with a gene or genes responsible for self-incompatibility (SI) in giant ragweed, and that linkage drag was disrupting pollination in resistant plants. This research provided evidence that giant ragweed does possess SI, as greater pollen retention, pollen tube growth, and seed set were observed in cross-pollinated plants compared with self-pollinated plants. Non-Mendelian inheritance of the Trp574Leu mutation was documented in crosses between plants from three different giant ragweed populations, indicating that the mutant <em>ALS</em> allele may be linked with an SI allele common to many plants because of a shared resistant ancestor.  In crosses between plants from one population, production of resistant F1 seeds was 33% greater on average compared with the expectation under Mendelian inheritance. </p> <p>Collectively, this research demonstrated that mesotrione may become a highly effective tool for control of giant ragweed in soybean. Applications of mesotrione can also reduce the selection for an increased frequency of ALS inhibitor-resistant biotypes induced by cloransulam, though a more robust weed management strategy may be necessary to maintain the long-term viability of ALS inhibitors.  The need for sound weed management practices is underscored by the impact of the linkage of SI and <em>ALS</em> genes, which may be encouraging a more rapid spread of herbicide-resistance than was previously anticipated.</p>