Regulation of dhurrin synthesis in forage sorghum

Sorghum is a C4 photosynthetic, drought resistant forage and grain plant species. Sorghum is cyanogenic in all tissues except for the mature seeds. The cyanogenesis pathway in sorghum produces the stable cyanogenic glucoside, dhurrin. The three biosynthetic genes in the cyanogenesis pathway are CYP79A1, CYP71E1 and UGT85B1. The first cytochrome P450, CYP79A1, is thought to be the rate limiting enzyme. Various sorghum lines were utilised for this study, with the mean hydrogen cyanide potential (HCNp) and coding sequence for CYP79A1 previously determined (O'Donnell et al., 2013). The coding sequences did not explain the observed difference in HCNp. Therefore, the 1.2Kb promoter region of CYP79A1 was sequenced and in silico analysis performed to look for a regulatory explanation to match the HCNp variances. Sequence variations were identified with potential cis-acting regulatory motifs exhibiting sequence alterations. When the cells of sorghum leaf tissue are masticated the dhurrin is hydrolysed by dhurrinase and hydrogen cyanide (HCN) is released. At low concentrations HCN can be metabolised safely by mammals but at higher concentrations it can cause toxic metabolic asphyxiation. The HCNp of sorghum is important in the cattle industry as forage sorghum is used as an alternate feed crop over summer. Many of the motifs identified in the CYP79A1 promoter region are potential light, circadian and/or temperature response elements. The regulation of dhurrin production has been investigated to determine if dhurrin is constitutively produced or whether the cyanogenesis pathway is under the control of a circadian or diurnal rhythm. Multiple experiments were conducted to compare dhurrin content under different light and temperature treatments in hydroponically grown forage sorghum and the results indicate that dhurrin is not under circadian or diurnal regulation. A gel mobility shift assay system was set up to look for DNA-binding proteins associating with the CYP79A1 promoter region. This method was successful in finding proteins that bound to fragments of the CYP79A1 promoter, which were then identified by isolating the proteins of interest and sending them for N-terminal sequencing at the Australian Proteome Analysis Facility (APAF). This resulted in the identification of a histone H1 protein and a histone H3 protein that strongly bound to a 62 base pair fragment of the CYP79A1 promoter, approximately 380 nucleotides upstream from the start codon. Histone H3 variants have been shown to be involved in transcriptional activation and the histones found could be part of a larger histone complex. This work provides a starting point for understanding the molecular mechanisms controlling the cyanogenesis biosynthesis pathway