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

Abstract

SummaryCyanogenic 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 wildtype 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.

Drought-induced changes in nitrogen partitioning between cyanide and nitrate in leaves and stems of sorghum grown at elevated CO2 are age dependent

“…Foliar dhurrin (cyanogenic glucoside) concentrations were determined on leaves (i.e., leaf blade) and stems (comprising both the stem and the enclosing leaf sheaths) using the evolved cyanide method following Blomstedt et al (2012). Exogenous ␤-glucosidase (1.12 units mL −1 ) from almond (EC 3.2.1.21, Sigma) was used to ensure that all dhurrin was converted to cyanide during incubation.…”

Lotus japonicus flowers are defended by a cyanogenic β-glucosidase with highly restricted expression to essential reproductive organs

“…Over 3000 different plant species have been reported to contain a-HNGs, with most studies and surveys concentrating on foliar tissues (Harper et al 1976;Conn 1981; Lewis and Zona 2000;Forslund et al 2004;Miller et al 2006;Blomstedt et al 2012;Miller and Tuck 2013;Neilson et al 2015) or the edible parts of food crops (Jones 1998;Nielsen et al 2002;Jørgensen et al 2005). There are relatively few reports of the occurrence of a-HNGs in flower tissues but in Grevillea species, Linum usitatissimum (flax), L. japonicus, Ryparosa kurrangii (rainforest tree) and Eucalyptus camphora they accumulate in equivalent or higher levels than foliar tissues (Lamont 1994;Niedźwiedź-Siegień 1998;Forslund et al 2004;Webber and Woodrow 2008;Neilson et al 2011).…”

Plant Desiccation Tolerance and its Regulation in the Foliage of Resurrection “Flowering-Plant” Species

“…Another powerful functional genetics methodology, which does not necessitate the development of DNA transformation technology, involves targeting induced local lesions in genomes (TILLING) [171,172]. This approach has been successfully used to identify mutants in monocotyledonous crops that possess disruptions in specific genes [172][173][174]. An alternative approach which has proven valuable and which may be of direct interest to agricultural crops, is the ectopic expression of genes from resurrection plants in otherwise desiccation-sensitive species where reproducible transformation protocols do exist.…”