POSSIBLE USE OF A BIOTECHNOLOGICALAPPROACH TO OPTIMIZE AND REGULATETHE CONTENT AND DISTRIBUTION OFCYANOGENIC GLUCOSIDES IN SORGHUM TOINCREASE FOOD SAFETYBowater/MiraImagesdailykos
Why CyanogenicGlucosidesas a Topic?• Cyanogenic Plants?• CyanogenicGlucosides belong to the class ofphytoanticipins• Mechanical disruption of plant tissue?
• What are CyanogenicGlucosides?• There are four type of linkages presentbetween glycone and aglycone:1. C-linkage/glycosidic bond, "nonhydrolysableby acids or enzymes"2. O-linkage/glycosidic bond3. N-linkage/glycosidic bond4. S-linkage/glycosidic bond• Toxicity of CyanogenicGlucosides1. Repel Herbivores2. Relationship between microorg3. Easy hosts for fungi and insects-easyinceptors of pathogensawakeandlivingYikrazuul et al. 2008
Focus on Sorghum(Sorghum bicolor L.)?• Naturally occuringacyanogenic individuals foundin cyanogenic plant species- white clover• Problem with Sorghum:
• Sorghum cyanogenicglucoside = dhurrin• Problem with dhurrin? ---hydrolysed by B-glucidosases• Sorghum as animal fodder-sorghum forage• Overall:
Plant Biotechnology --- please help!Peter Stuart et al. 2012Robyn OBrien et al. 2012There is a great need for acyogenic forage production
• Understand the regulation of dhurrin content in sorghumseedlings. dhurrin synthesis in sorghum seedlings is regulated bythe rate of de novo synthesis of the biosynthetic enzymes.• Dhurrin synthesis: involves two cytochrome P450s (CYP79A1 andCYP71E1) and one UDP-glucosyltransfer- ase (UGT85B1)
• Peter Kamp Busk2 and Birger LindbergMøller* Studies on Cyanide Potential• the activity of the first enzyme in the pathwayis always rate limiting• Outlook: CYP79A1 mutations• TILLING for forage sorghum:Cecilia K. Blomstedt et al. 2011
• P414L mutation in CYP79A1Cecilia K. Blomstedt et al. 2011
How did we get to the P414L mutation?Cecilia K. Blomstedt et al. 2011
• Cytochrome P450’s highly substrate specific• P414L mutation decreases substrate affinity• Normal: E-R-R triad: arginine (R) in PERF motif and arginineresidues (R) and glutamic acid (E) in KETLR motif• locks the haem pocket of active site into proper position• P141L mutationProsser et. al. 2006
References• Blomstedt, C. K., Gleadow, R. M., ODonnell, N., Naur, P., Jensen, K., Laursen, T., & Olsen, C. E.(2012). A combined biochemical screen and TILLING approach identiﬁes mutations in Sorghumbicolor L. Moench resulting in acyanogenic forage production. Plant Biotechnology Journal, 10, 54-66.• Busk, P. K., &Moller, B. L. (2002, July). Dhurrin Synthesis in Sorghum Is Regulated at theTranscriptional Level and Induced by Nitrogen Fertilization in Older Plants. Plant Physiology, 129,1222-1231.• Ganjewala, D., Kumar, S., S, A. D., &Ambika, K. (2010). Advances in cyanogenic glycosidesbiosynthesis and analyses in plants: A review. ActaBiologicaSzegediensis, 54(1), 1-14.• Halkier, B. A., &Moller, B. L. (1990, June 18). The biosynthesis of cyanogenicglucosides in higherplants. The journal of biological chemistry, 54(1), 21114-21121.• Prosser, D. E., YuDing, G., Zongchao, J., & Glenville, J. (2006, May). Structural motif-based homolgymodeling of CYP27A1 and site-directed mutational analyses affecting vitamin D hydroxylation.Biophysical Journal, 90(10), 3389-3409.• Trigiano, R. N., Windham, M. T., & Windham, A. S. (2003). Plant pathology concepts and laboratoryexercizes. CRC Press, 447.• Wheeler, J. L., &Mulcahy, C. (1989, December). Consequences for animal production ofcyanogenesis in sorghum forage and hay. Tropical Grasslands, 23(4), 21114-21121.