Regulation of benzylisoquinoline alkaloid biosynthesis in opium poppy

The transcriptional regulation of benzylisoquinoline alkaloid biosynthesis is investigated in opium (Papaver somniferum L.) and California (Eschscholzia californica Cham.) plants. Using a combination of functional analysis of the P. somniferum promoter in the E. californica heterologous host, yeast one-hybrid assays using P. somniferum elicited cell cultures and roots as prey and a bioinformatics-phylogenetic approach the regulation of BIA biosynthesis is investigated.;Arbitrarily selected 200 by segments making up the first 600 by of the P. somniferum BBE promoter were manipulated for use as bait in a yeast one-hybrid system. This bait was screened against a GAL4-transactivation domain-cDNA fusion library using P. somniferum elicited cell culture and root RNA as the source for the library. The yeast one-hybrid screen, while yielding no positive DNA binding clones to date can now be optimized given the data within to increase the possibility of identifying a trans-acting factor regulating BIA biosynthesis. A tool for use as a bait positive control in this system has also begun to be developed, which may be useful given that currently no DNA elements have been delineated to less than ∼150 by to date in this system. We have also begun to examine the possibility of using software predicted genetic elements and the whole sequence of the P. somniferum BBE promoter for use as bait in the yeast one-hybrid screen.;The use of genomics tools, promoter prediction programs and phylogenetic analysis of putative clones involved as transcription factors regulating BIA biosynthesis has allowed identification of a small subset of ESTs which are now significant candidates for functional analysis to confirm their role in P. somniferum. EST sequences of interest as putative regulators of BIA biosynthesis were identified first by examining their expression patterns using a microarray approach (information supplied by K. Zulak). Correlation of this with predicted binding sites in the P. somniferum BBE promoter and with phylogenetic analyses of families of transcription factors representing these clones of interest has allowed us to focus further on a subset of these ESTs for initial characterization, potentially increasing our ability to identify a regulator of BIA biosynthesis with fewer functional assays.;Finally, the viability of other members of the Ranunculaceae to induction of hairy root cultures using A. rhizogenes R1000 has allowed us to identify a number of species with alkaloid profiles unique from the current species being used as models for BIA biosynthesis. Given the relative ease with which some of these species were able to regenerate hairy roots it may be possible to use these species in other transformation protocols in the future, expanding our tools to investigate the evolution of BIA biosynthesis and possibly introducing species as targets for metabolic engineering in the future.;A series of deletions of the full length of the P. somniferum BBE promoter driving expression of two variations on the GUSa gene reporter were examined in terms of their patterns and levels of expression in E. californica hairy root cultures using A. rhizogenes R1000. This functional analysis of the P. somniferum BBE promoter in E. californica has lead to a better understanding of which sections of the promoter are involved in expression in this heterologous host.