<p>Dhurrin is a cyanogenic glucoside (CG), an important compound that can interplay with primary and secondary metabolism in sorghum. Dhurrin metabolism contributes to insect resistance, growth, nitrogen (N) metabolism, drought tolerance, and safety for animal consumption when used as a forage. Through chemical mutagenesis with ethyl methanesulfonate (EMS), a mutation in the gene encoding CYP79A1 (<i>cyp79a1</i>), the first enzyme in the biosynthetic pathway of dhurrin, was discovered that inhibits the production of dhurrin. The acyanogenic phenotype of this mutant could be a major benefit in reducing the risk of hydrogen cyanide (HCN) toxicity within animals; however, understanding the effects of inhibiting dhurrin biosynthesis is important in understanding metabolic tradeoffs that could occur. This dissertation describes research to assess impacts and tradeoffs of the dhurrin-free trait on susceptibility to Fall Armyworm [<i>Spodoptera frugiperda</i> (J.E. Smith)] (FAW) feeding, seedling growth, effects on post-flowering drought tolerance, cold stress and utilization as a forage. Insect susceptibility and seedling growth were examined using near-isogenic lines (NILs) within the greenhouse utilizing non-destructive phenotyping technologies for green plant area and in the field comparing total leaf area and dry weight. Post-flowering drought stress was induced within a greenhouse, growth chamber, and field environments. The <i>cyp79a1 </i>mutation was tested in NILs, a near-isogenic backcross (NIBC) population, and near-isogenic hybrids (NIH), to understand the impacts of the <i>cyp79a1 </i>mutation<i> </i>on the stay-green trait. Palatability as forage was examined by comparing the feeding preference of ruminant animals with multiple conventional hybrids and an experimental hybrid carrying the <i>cyp79a1</i> mutation. This preference was also examined using a set of NILs varying in the <i>cyp79a1 </i>mutation<i>. </i> Safety was assessed in preference trials by testing for HCN release before grazing. To further our understanding of the benefits of sorghum as a forage, the dhurrin-free experimental hybrid was compared to seven conventional hybrids as a dry product. The dry sorghum product was tested for the release of HCN and dhurrin content. Lastly, the effects of low temperatures and frost were assessed for their effects on the production of dhurrin in cyanogenic and dhurrin-free sorghum genotypes. </p> <p>Overall, the biosynthesis of dhurrin had a significant effect on the deterrence of FAW and on the growth of sorghum seedlings. Dhurrin-free lines were more susceptible to FAW feeding but also exhibited a significantly higher growth rate. Dhurrin-free lines and hybrids only exhibited a slight increase in susceptibility to post-flowering drought stresses with only one dhurrin-free hybrid discovered to senesce faster than its wild-type NIH. Comparisons of the effects of dhurrin biosynthesis on stay-green in a NIBC population in Tx642 (B35), one of the most important sources of the stay-green trait, did not show any variation in chlorophyll concentration (CC) and normalized difference vegetation index (NDVI). Analyses of the impact of dhurrin on palatability as a forage showed that ewes preferred grazing on the dhurrin-free hybrids and NILs, showing that the ewes were able to detect the presence or absence of dhurrin while feeding. Experiments to assess the safety and stability of dhurrin in dried plant material demonstrated that dhurrin content did not change during drying and HCN was released after rehydration. Furthermore, high levels of HCN were immediately released when rumen fluid was added to dried plant materials containing dhurrin; however, no detectable HCN was released from dhurrin-free genotypes. Finally, sorghum plants exposed to freezing temperatures exhibited an increase in dhurrin content in conventional sorghum hybrids while no detectable dhurrin was noted within <i>cyp79a1 </i>mutants. </p> <p>Taken together, these studies demonstrate pleiotropic effects for the <i>cyp79a1 </i>mutation. Dhurrin-free genotypes were more susceptible to insect herbivory and may be slightly more susceptible to post-flowering drought within the hybrids; however, these genotypes exhibited higher seedling growth rates, feeding preference by ewes, no release of HCN in fresh or dry plant material, and frost did not cause an increase in dhurrin content.</p>

β-glucosidases catalyze the hydrolysis of aryl and alkyl β-D-glucosides as well as glucosides with a carbohydrate moiety. The maize β-glucosidase isozymes Glu1and Glu2 hydrolyze a broad spectrum of substrates in addition to its natural substrate DIMBOAGlc, while the sorghum β-glucosidase Dhr1 (dhurrinase-1) hydrolyzes exclusively its natural substrate dhurrin. For the expression of mature β-glucosidase isozymes Glu1 and Glu2 of maize and Dhr1 of sorghum, complementary DNAs were amplified by PCR and cloned into the expression vector pET-21a. Recombinant Glu1, Glu2 and Dhr1 enzymes were found to display activity towards the physiological substrates DIMBOAGlc and dhurrin, respectively, at levels similar to their native counterparts. It has been a subject of the subsequent studies by our lab and others to investigate what determines the aglycone specificity in β-glucosidases, and how β-glucosidases catalyze the hydrolysis of β-glycosidic bond between sugar and aglycone moieties. Molecular modeling techniques allowed to predict the substrate binding sites in Glu1 and Dhr1. Based on structural analysis of Glu1 and Dhr1, chimeric β-glucosidases (Glu1/Dhr1) were constructed by shuffling the C-terminal amino acids of Glu1 with the homologous region of Dhr1 to study the mechanism of substrate specificity. The resulting chimeric enzymes were characterized with respect to substrate specificity as well as kinetic, immunological, and electrophoretic properties. Shuffling a small portion of the C-terminal region altered the substrate specificity and improved by 2-4 fold the catalytic efficiency on other substrates in the chimeric β-glucosidases. These experiments showed that one or more of the 10 amino acid substitutions in the 30 amino acid long Dhr1 subdomain, 462SSGYTERFGIVYVDRENGCERTMKRSARWL491, plays a key role in dhurrin recognition and hydrolysis. To further investigate dhurrin recognition within this peptide region, two chimeric enzymes containing ⁴⁶²SSGYTERF⁴⁶⁹ and ⁴⁶⁶FAGFTERY⁴⁷³ Dhr1 peptides, respectively, were generated. The kinetic parameters indicated that Dhr1 peptide, ⁴⁶²SSGYTERF⁴⁶⁹, alone is sufficient to convert Glu1 to Dhr1 substrate specificity when it replaces the homologous peptide, ⁴⁶⁶FAGFTERY⁴⁷³, of Glu1. Maize β-glucosidases share high sequence similarities with Family 1 O-glucosidases. Therefore, these proteins are classified as retaining glycosyl hydrolases whose active site contains two glutamic acids (E) as the key catalytic residues, one as a general acid/base catalyst (E191) and the other as a nucleophile (E406). To confirm the identity and function of the acid/base catalyst E191, we have changed this residue to isosteric glutamine (Q) and aspartic acid (D) in both Glu1 and Glu2 isozymes by site-directed mutagenesis. The resulting mutant proteins were purified and their kinetic parameters (K_m, k_cat and k_i) were determined. The replacement of the acid/base catalyst E191 in the active site of maize β-glucosidase by Q and D resulted in inactivation of the enzyme. The kinetic analysis of the E191Q mutants showed that catalytic activity was reduced 200- and 110-fold towards ortho- and para-nitrophenyl- β-D-glucosides, respectively, when compared with the wild type enzyme. The E191D mutants showed no detectable activity towards any of the substrates tested. The back mutation of the E191Q mutants of the Glu1 and Glu2 isozymes to wild type restored full catalytic activity in both cases. These data indicate that E191 in maize β-glucosidases functions as an acid/base catalyst, and its function in catalysis cannot be performed by an isosteric residue such as glutamine or by a carboxyl group on a shorter side chain such as in aspartic acid. / Ph. D.