A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production

• Published in: Plant biotechnology journal Vol. 10; no. 1; pp. 54 - 66
• Main Authors: Blomstedt, Cecilia K., Gleadow, Roslyn M., O'Donnell, Natalie, Naur, Peter, Jensen, Kenneth, Laursen, Tomas, Olsen, Carl Erik, Stuart, Peter, Hamill, John D., Møller, Birger Lindberg, Neale, Alan D.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.01.2012; Blackwell
• Subjects: Animal Feed; Animals; Biological and medical sciences; biosynthesis; Biosynthetic Pathways; Biotechnology; Biotechnology - methods; Blotting, Western; Crosses, Genetic; cyanide toxicity; CYP79A1; cytochrome P-450; Cytochrome P-450 Enzyme System; Cytochrome P-450 Enzyme System - genetics; enzymes; enzymology; Ethyl Methanesulfonate; forage production; Fundamental and applied biological sciences. Psychology; gene regulation; genes; genetic techniques and protocols; genetics; Genome, Plant; Genome, Plant - genetics; glucosides; Glycosides; Glycosides - metabolism; Humans; hydrogen cyanide; Hydrogen Cyanide - metabolism; leaves; mature plants; metabolism; methods; Microsomes; Microsomes - enzymology; Models, Molecular; molecular models; Mutagenesis; Mutagenesis - genetics; mutants; mutation; Mutation - genetics; mutations; NADP; NADP - metabolism; Nitriles; Nitriles - metabolism; nitrogen metabolism; Phenotype; screening; seedlings; seeds; Sorghum; Sorghum - enzymology; Sorghum - genetics; Sorghum bicolor; Structural Homology, Protein; Plant production; Monocotyledones; Toxicity; nitrogen metabolism; Biochemistry; Cyanides; Nitrogen; Gene expression; Metabolism; CYP79A1; Regulation(control); Sorghum bicolor; mutations; Gene; Gramineae; gene regulation; cyanide toxicity; Angiospermae; Spermatophyta; Mutation; Fodder crop
• ISSN: 1467-7644; 1467-7652; 1467-7652
• DOI: 10.1111/j.1467-7652.2011.00646.x

Summary Cyanogenic glucosides are present in several crop plants and can pose a significant problem for human and animal consumption, because of their ability to release toxic hydrogen cyanide. Sorghum bicolor L. contains the cyanogenic glucoside dhurrin. A qualitative biochemical screen of the M2 population derived from EMS treatment of sorghum seeds, followed by the reverse genetic technique of Targeted Induced Local Lesions in Genomes (TILLING), was employed to identify mutants with altered hydrogen cyanide potential (HCNp). Characterization of these plants identified mutations affecting the function or expression of dhurrin biosynthesis enzymes, and the ability of plants to catabolise dhurrin. The main focus in this study is on acyanogenic or low cyanide releasing lines that contain mutations in CYP79A1, the cytochrome P450 enzyme catalysing the first committed step in dhurrin synthesis. Molecular modelling supports the measured effects on CYP79A1 activity in the mutant lines. Plants harbouring a P414L mutation in CYP79A1 are acyanogenic when homozygous for this mutation and are phenotypically normal, except for slightly slower growth at early seedling stage. Detailed biochemical analyses demonstrate that the enzyme is present in wild‐type amounts but is catalytically inactive. Additional mutants capable of producing dhurrin at normal levels in young seedlings but with negligible leaf dhurrin levels in mature plants were also identified. No mutations were detected in the coding sequence of dhurrin biosynthetic genes in this second group of mutants, which are as tall or taller, and leafier than nonmutated lines. These sorghum mutants with reduced or negligible dhurrin content may be ideally suited for forage production.