Metabolic consequences of knocking out UGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin in Sorghum bicolor (L. Moench (original) (raw)
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Plant Biotechnology 10:54-66
Cyanogenic glucosides are present in several crop plants and can pose a significant problem for human and animal consumption, due to their ability to release toxic hydrogen cyanide. Sorghum bicolor L. contains the cyanogenic glucoside dhurrin. A biochemical screen and the reverse genetic technique of Targeted Induced Local Lesions in Genomes (TILLING) were employed to identify mutants with altered hydrogen cyanide potential (HCNp). Characterisation 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 paper 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. 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 non-mutated lines. The sorghum mutants with reduced or negligible dhurrin content that have been produced may be ideally suited for forage production.
Cyanogenesis in the Sorghum Genus: From Genotype to Phenotype
Genes
Domestication has resulted in a loss of genetic diversity in our major food crops, leading to susceptibility to biotic and abiotic stresses linked with climate change. Crop wild relatives (CWR) may provide a source of novel genes potentially important for re-gaining climate resilience. Sorghum bicolor is an important cereal crop with wild relatives that are endemic to Australia. Sorghum bicolor is cyanogenic, but the cyanogenic status of wild Sorghum species is not well known. In this study, leaves of wild species endemic in Australia are screened for the presence of the cyanogenic glucoside dhurrin. The direct measurement of dhurrin content and the potential for dhurrin-derived HCN release (HCNp) showed that all the tested Australian wild species were essentially phenotypically acyanogenic. The unexpected low dhurrin content may reflect the variable and generally nutrient-poor environments in which they are growing in nature. Genome sequencing of six CWR and PCR amplification of th...
Cyanogenic Glycosides: Synthesis, Physiology, and Phenotypic Plasticity
Cyanogenic glycosides (CNglcs) are bioactive plant products derived from amino acids. Structurally, these specialized plant compounds are characterized as α-hydroxynitriles (cyanohydrins) that are stabilized by glucosylation. In recent years, improved tools within analytical chemistry have greatly increased the number of known CNglcs by enabling the discovery of less abundant CNglcs formed by additional hydroxylation, glycosylation, and acylation reactions. Cyanogenesis—the release of toxic hydrogen cyanide from endogenous CNglcs—is an effective defense against generalist herbi-vores but less effective against fungal pathogens. In the course of evolution, CNglcs have acquired additional roles to improve plant plasticity, i.e., establishment , robustness, and viability in response to environmental challenges. CNglc concentration is usually higher in young plants, when nitrogen is in ready supply, or when growth is constrained by nonoptimal growth conditions. Efforts are under way to engineer CNglcs into some crops as a pest control measure, whereas in other crops efforts are directed toward their removal to improve food safety. Given that many food crops are cyanogenic, it is important to understand the molecular mechanisms regulating cyanogen-esis so that the impact of future environmental challenges can be anticipated.
Planta
Main conclusion Australian native species of sorghum contain negligible amounts of dhurrin in their leaves and the cyanogenesis process is regulated differently under water-stress in comparison to domesticated sorghum species. Abstract Cyanogenesis in forage sorghum is a major concern in agriculture as the leaves of domesticated sorghum are potentially toxic to livestock, especially at times of drought which induces increased production of the cyanogenic glucoside dhurrin. The wild sorghum species endemic to Australia have a negligible content of dhurrin in the above ground tissues and thus represent a potential resource for key agricultural traits like low toxicity. In this study we investigated the differential expression of cyanogenesis related genes in the leaf tissue of the domesticated species Sorghum bicolor and the Australian native wild species Sorghum macrospermum grown in glasshouse-controlled water-stress conditions using RNA-Seq analysis to analyse gene expression. The ...
BMC genomics, 2016
The important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed. In this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. Dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. During the subsequent maturation period, the dhurrin content was turned over, resulting in only negligible residual dhurrin amounts in the mature grain. Dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. Despite the accumulation of dhurrin, the grains were acyanogen...
The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways
The Plant journal : for cell and molecular biology, 2015
The biosynthetic pathway for the cyanogenic glucoside, dhurrin, in sorghum has previously been shown to involve the sequential production of (E) - and (Z)-p-hydroxyphenylacetaldoxime. In this study we used microsomes prepared from wild type and mutant sorghum or transiently transformed Nicotiana benthamiana to demonstrate that CYP79A1 catalyzes conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime whereas CYP71E1 catalyzes conversion of (E)-p-hydroxyphenylacetaldoxime into the corresponding geometrical Z-isomer as required for its dehydration into a nitrile, the next intermediate in cyanogenic glucoside synthesis. Glucosinolate biosynthesis is also initiated by the action of a CYP79 family enzyme but the next enzyme involved belongs to the CYP83 family. We demonstrate that CYP83B1 from Arabidopsis thaliana cannot convert the E-oxime to the Z-isomer, which blocks the route towards cyanogenic glucoside synthesis. Instead CYP83B1 catalyzes the conversion of the E-oxime into an S-al...