Regulation of cyanogenesis in forage sorghum
thesisposted on 07.12.2016 by O'Donnell, Natalie Hélène
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The C4 plant Sorghum combines high water use efficiency with heat tolerance making it suitable as a fodder crop in the dry tropics. However, it also produces dhurrin (Conn 1981) which leads to the release of hydrogen cyanide (HCN) when plants are damaged. The HCN potential (HCNp) of sorghum is subject to spatial and temporal regulation and levels can vary greatly between varieties (Benson et al. 1969; Haskins et al. 1987). Sorghum HCNp is also affected by environmental factors including drought, frost and soil fertility, and levels within a given variety can differ from one season to the next (Gray et al. 1968). Experienced farmers know that young sorghum plants, or older plants that have been subjected to drought, are often toxic to grazing cattle. Three key genes catalyse the biosynthesis of dhurrin in sorghum, two cytochrome P450s (CYP79A1 and CYP71E1) and a (UDP)-glycosyltransferase (UGT85B1). The CYP79A1, CYP71E1 and UGT85B1 enzymes, along with the redox partner NADPH-dependant cytochrome P450 reductase (CPR) (Ellis et al. 2009; Jensen & Møller 2010), are thought to form a metabolon to enable channelling of toxic intermediates (Nielsen et al. 2008). CYP79A1 is thought to be the rate limiting step in the pathway, but little more is known about the regulation of these genes. It has been hypothesised in numerous studies that stress, in particular osmotic and nutrient related stress, play a large role in the regulation of HCNp in sorghum. However, it is not known how these factors influence the cyanogenesis pathway. The overall aim of this project was to generate new varieties of sorghum with lower cyanogenic glucoside content for safer consumption by cattle, particularly after drought. Within this larger project the specific aims of this PhD project were to (1) to determine the ontogenetic changes in the HCNp of forage sorghum, (2) to determine the effect of environmental stress on dhurrin concentration, and (3) to identify a viable low dhurrin producing sorghum line. The variation in HCNp due to ontogenic changes was investigated which confirmed that the HCNp of sorghum is highest in seedlings and decreases with plant age. However, in sorghum the total dhurrin content increases with age. Numerous abiotic and biotic stresses (osmotic, high/low nitrogen, wounding and different hormone treatments) were applied to the plants to monitor the effect on accumulation of dhurrin in various parts of sorghum plants. Results showed that the dhurrin concentration increased in the shoots under osmotic stress, high nitrogen (plants >6 leaves), and when treated with the hormones Methyl jasmonate, Kinetin and Abscisic acid. In general the different treatments did not induce HCNp in the roots. The transcript level of CYP79A1, the rate limiting step in dhurrin biosynthesis, was analysed by quantitative RT-PCR (qPCR) and an inverse relationship was found between transcript levels and HCNp in leaf sheath tissue. The relationship between CYP79A1 transcript levels and HCNp in the leaf blade and roots was not as defined and will require further detailed investigation. Evidence presented in this thesis suggests that the change in dhurrin concentration in sorghum may be due to a reduction in plant growth caused by environmental stresses, leading to a feedback loop that inhibits the turnover of dhurrin, rather than an increase in activity of key biosynthetic genes per se. Targetted Induced Local Lesions in Genomes (TILLING) and cyanide assays identified several mutant lines with altered HCNp. Detailed characterisation of an acyanogenic line, termed total cyanide deficient (tcd2), identified the mutation as being in the UGT85B1 gene resulting in a stop codon. The tcd2 line is novel because it was previously thought that a mutation the UGT85B1 gene would result in the accumulation of toxic intermediates and therefore a non viable plant. However growth studies found other than a stunted phenotype and a small delay in development, the tcd2 line is fully functional and fertile. The tcd2 line is now being used to further characterise the putative dhurrin turnover pathway. The findings detailed in this thesis have wide implications for further characterisation of the molecular mechanisms that regulate dhurrin synthesis in sorghum as well as practical applications for farmers in to agricultural regions experiencing environmental stress due to climate change. The cattle industry that use forage sorghum as fodder will benefit from an understanding of the regulation of cyanogenesis in sorghum. Additionally, the information gained on the regulation of cyanogenesis will have worldwide implications as it may be transferable to other cyanogenic crops which are important food resources, such as cassava.