THE EFFECTS OF INHIBITING DHURRIN BIOSYNTHESIS IN SORGHUM
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 (cyp79a1), 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 [Spodoptera frugiperda (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 cyp79a1 mutation was tested in NILs, a near-isogenic backcross (NIBC) population, and near-isogenic hybrids (NIH), to understand the impacts of the cyp79a1 mutation 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 cyp79a1 mutation. This preference was also examined using a set of NILs varying in the cyp79a1 mutation. 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.
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 cyp79a1 mutants.
Taken together, these studies demonstrate pleiotropic effects for the cyp79a1 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.
Collaborative Research: NRT-INFEWS: Sustainable Food, Energy, and Water Systems (SFEWS)
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