Borage (Borago officinalis) is an annual herb plant for culinary and medicinal uses. Due to a high level of gamma-linolenic acid (GLA) in its seed oil and the health-related benefits of GLA, borage is commercially cultivated. However, a herbicide-resistant variety has not yet been developed for effective weed management in borage farming. Thus, this thesis aimed to create, identify and characterize ethyl methanesulfonate (EMS) induced borage mutants for herbicide imidazolinone resistance. An EMS-mutagenized borage population was generated by using a series of concentrations of EMS to treat M1 seeds. After screening M2 borage plants with the herbicide, tolerant plants were selected, self-pollinated and grown to their maturity. The offsprings were subjected to herbicide screening again to confirm the phenotype, resulting in identification of two genetically stable imidazolinone-resistant lines. Two acetohydroxyacid synthase (AHAS) genes, AHAS1 and AHAS2, involved in the imidazolinone resistance were isolated and sequenced from both mutant (resistant) and wild type (susceptible) borage plants. Comparison of these AHAS sequences revealed that a single nucleotide substitution occurred in the AHAS1 resulting in an amino acid change from serine (S) in the susceptible plant to asparagine (N) in the first resistant line. The similar substitution was later found in the AHAS2 of the second resistant line. A KASP marker was developed for the AHAS1 mutation to differentiate the homozygous susceptible, homozygous and heterozygous resistant borage plants for the breeding purpose. The in vitro assay showed homozygous resistant borage containing the AHAS1 mutation could retain significantly higher AHAS activity than susceptible borage across different imazamox concentrations. The herbicide dose response test showed that the resistant line with the AHAS1 mutation was tolerant to four times the field applied concentration of the “Solo” herbicide.

Doctor of Philosophy / Department of Agronomy / Kassim Al-Khatib / Mitchell R. Tuinstra / Sorghum acreage is declining throughout the United States because management options and yield have not maintained pace with maize improvements. The most extreme difference has been the absence of herbicide technology development for sorghum over the past twenty years. The objectives of this study were to evaluate the level of resistance, type of inheritance, and causal mutation of wild sorghums that are resistant to either acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides or acetohydroxyacid synthase (AHAS)-inhibiting herbicides. ACCase-inhibiting herbicides used in this study were aryloxyphenoxypropionate (APP) family members fluazifop-P and quizalofop-P along with cyclohexanedione (CHD) family members clethodim and sethoxydim. The level of resistance was very high for APP herbicides but low to nonexistent to CHD herbicides. With genetic resistance to APP herbicides, the resistance factors, the ratio of resistance to susceptible, were greater than 54 to 64 for homozygous individuals and greater than 9 to 20 for heterozygous individuals. Resistance to CHD herbicides was very low with resistance factors ranging from one to about five. Genetic segregation studies indicate a single gene is the cause of resistance to APP herbicides. Sequencing identified a single mutation that results in cysteine replacing tryptophan (Trp-2027-Cys). Trp-2027-Cys has previously been reported to provide resistance to APP but not CHD herbicides. The other wild sorghum evaluated in this study was resistant to AHAS-inhibiting herbicides including imidazolinone (IM) family member, imazapyr, and sulfonylurea (SU) family member, nicosulfuron. Resistance factors in this genotype were very high, greater than 770 for the IM herbicide and greater than 500 for the SU herbicide, for both herbicide chemical families. Genetic segregation studies demonstrate that resistance was controlled by one major locus and two modifier loci. DNA sequencing of the AHAS gene identified two mutations, Val-560-Ile and Trp-574-Leu. Val-560-Ile is of unknown importance, but valine and isoleucine are similar and residue 560 is not conserved. Trp-574 is a conserved residue and Leu-574 is a known mutation that provides strong cross resistance to IM and SU herbicides. The results of these studies suggest that these sources of APP, SU, and IM resistance may provide useful herbicide resistance traits for use in sorghum.

Acetohydroxyacid synthase is a multisubunit enzyme that catalyses the first committed step in the biosynthesis of the branched chain amino acids viz., valine, leucine and isoleucine. In order to understand the structural basis for the observed allosteric feedback inhibition in AHAS, the regulatory subunit of AHAS isozymes I from E. coli was cloned, expressed, purified and the conditions were optimized for solution NMR spectroscopy. IlvN was found to exist as a dimer both in the presence and absence of the feedback inhibitor. Using high-resolution multidimensional, multinuclear NMR experiments, the structure of the dimeric valine-bound 22 kDa IlvN was determined. The ensemble of twenty low energy structures shows a backbone root mean square deviation of 0.73 ± 0.13 Å and a root mean square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, greater than 98% of the backbone φ, ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map. Each protomer exhibits a βαββαβα topology that is a characteristic feature of the ACT domain fold that is observed in regulatory domains of metabolic enzymes. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR timescale. Important structural properties of the unliganded state were probed by H-D exchange studies by NMR, alkylation studies by mass spectrometry and other biophysical methods. It was observed that the dynamic unliganded IlvN underwent a coil-to-helix transition upon binding the effector molecule and this inherent conformational flexibility was important for activation and valine-binding. A mechanism for allosteric regulation in the AHAS holoenzyme was proposed. Study of the structural and conformational properties of IlvN enabled a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis. Solution structural studies of 32 kDa Protease-VPg (PVPg) from Sesbania mosaic virus (SeMV) Polyprotein processing is a commonly found mechanism in animal and plant viruses, by which more than one functional protein is produced from the same polypeptide chain. In Sesbania Mosaic Virus (SeMV), two polyproteins are expressed that are catalytically cleaved by a serine protease. The VPg protein that is expressed as a part of the polyprotein is an intrinsically disordered protein (by recombinant expression) that binds to various partners to perform several vital functions. The viral protease (Pro), though possessing the necessary catalytic residues and the substrate binding pocket is unable to catalyse the cleavage reactions without the VPg domain fused at the C-terminus. In order to determine the structural basis for the aforementioned activation of protease by VPg I undertook the structural studies of the 32 kDa PVPg domains of SeMV by solution NMR spectroscopy. NMR studies on this protein were a challenge due to the large size and spectral overlap. Using a combination of methods such as deuteration, TROSY-enhanced NMR experiments and selective ‘reverse-labelling’, the sequence specific assignments were completed for ~80% of the backbone and 13C nuclei. NMR studies on mutants such as the C-terminal deletion mutant, I/L/V to A mutants in VPg domain were conducted in order to identify the residues important for aliphatic-aromatic interactions observed in PVPg. Attempts were made to obtain NOE restraints between Pro and VPg domains through ILV labelled samples; however these proved unsuccessful. It was observed that ‘natively unfolded’ VPg possessed both secondary and tertiary structure in PVPg. However, 30 residues at the C-terminus were found to be flexible. Even though atomic-resolution structure could not be determined, the region of interaction between the domains was determined by comparing NMR spectra of Pro and PVPg. The conditions for reconstitution of the Protease-VPg complex by recombinantly expressed Pro and VPg proteins were standardised. These studies lay the foundation for future structural investigations into the Protease-VPg complex.