POSSIBLE USE OF A BIOTECHNOLOGICAL

APPROACH TO OPTIMIZE AND REGULATE

THE CONTENT AND DISTRIBUTION OF

CYANOGENIC GLUCOSIDES IN SORGHUM TO

INCREASE FOOD SAFETY

Bowater/MiraImagesdailykos

Why CyanogenicGlucosidesas a Topic?

• Cyanogenic Plants?

• CyanogenicGlucosides belong to the class of phytoanticipins

• Mechanical disruption of plant tissue?

• What are CyanogenicGlucosides?

• There are four type of linkages present between 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 CyanogenicGlucosides

1. Repel Herbivores2. Relationship between microorg3. Easy hosts for fungi and insects-easy

inceptors of pathogens

awakeandliving

Yikrazuul et al. 2008

Focus on Sorghum (Sorghum bicolor L.)?

• Naturally occuringacyanogenic individuals found in 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. 2012

Robyn O'Brien et al. 2012

There is a great need for acyogenic forage production

• Understand the regulation of dhurrin content in sorghum seedlings. dhurrin synthesis in sorghum seedlings is regulated by the rate of de novo synthesis of the biosynthetic enzymes.

• Dhurrin synthesis: involves two cytochrome P450s (CYP79A1 and CYP71E1) and one UDP-glucosyltransfer- ase (UGT85B1)

Peter Kamp Busk et al. 2002

Kristensen et al., 2005; Jenrich et al., 2007

Kristensen et al., 2005; Jenrich et al., 2007

Kristensen et al., 2005; Jenrich et al., 2007

• Peter Kamp Busk2 and Birger Lindberg Møller* Studies on Cyanide Potential

• the activity of the first enzyme in the pathway is always rate limiting

• Outlook: CYP79A1 mutations

• TILLING for forage sorghum:

Cecilia K. Blomstedt et al. 2011

• P414L mutation in CYP79A1

Cecilia K. Blomstedt et al. 2011

How did we get to the P414L mutation?

Cecilia K. Blomstedt et al. 2011

Cecilia K. Blomstedt et al. 2011

Brandenburg et al. 2004

• Cytochrome P450’s highly substrate specific• P414L mutation decreases substrate affinity• Normal: E-R-R triad: arginine (R) in PERF motif and arginine

residues (R) and glutamic acid (E) in KETLR motif• locks the haem pocket of active site into proper position• P141L mutation

Prosser et. al. 2006

References

• Blomstedt, C. K., Gleadow, R. M., O'Donnell, N., Naur, P., Jensen, K., Laursen, T., & Olsen, C. E. (2012). A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor 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 the Transcriptional 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 glycosides biosynthesis 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 higher plants. 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 laboratory exercizes. CRC Press, 447.

• Wheeler, J. L., &Mulcahy, C. (1989, December). Consequences for animal production of cyanogenesis in sorghum forage and hay. Tropical Grasslands, 23(4), 21114-21121.