Regulation of Benzylisoquinoline Alkaloid Metabolism in Opium Poppy

Papaver somniferum, commonly known as opium poppy, is the primary source of opiate production, as well as the source of precursors to many semi-synthetic opioids used in the pharmaceutical industry. The array of alkaloids produced by P. somniferum results from the benzylisoquinoline alkaloid biosynthesis (BIA) pathway. The genes encoding the enzymatic steps involved in BIA metabolism have largely been elucidated and characterised; however, transcriptional regulation of this pathway remains to be understood. A forward genetics screen of mutagenised HN4 (high morphine and noscapine P. somniferum cultivar) identified two independent ‘loss of all alkaloids’ mutants: Δrba-1 and Δrba-2. Genome sequencing identified an overlapping region in both deletions containing a basic helix-loop helix transcription factor (bHLH TF), designated REGULATOR OF BENZYLISOQUINOLINE ALKALOIDS (RBA).
RNAseq identified a global downregulation of BIA biosynthetic genes in the Δrba-1 and Δrba-2 deletion mutants as a result of the RBA deletion. This Master’s thesis aimed to establish if Agrobacterium-mediated transformation of P. somniferum with a constitutively expressing CaMV-35S::RBA transgene would rescue the Δrba-1 and Δrba-2 mutant phenotype. Metabolite profiling of the 35S::RBA transformed mutants found that CaMV-35S::RBA results in a full rescue of the mutant phenotype: the complete restoration of noscapine and morphine accumulation. RNAseq analysis shows the addition of the 35S::RBA transgene to Δrba-2 restores the global gene expression profile to wildtype (WT) levels. Overexpressing RBA in the presence of a functional native RBA (WT + 35S::RBA) has no additional effect on metabolite accumulation or global gene expression.
In summary, constitutive RBA expression does rescue the Δrba mutant phenotype but is unable to drive alkaloid production beyond wild type levels. These findings establish RBA as a key transcription factor in BIA metabolism, providing insight into the regulatory control of specialised metabolism in P. somniferum.