Plant cells are highly compartmentalized with membranes that delineate organelles and provide physical barriers against movement of proteins and compounds. Many metabolic pathways span subcellular compartments, which necessitates transport of pathway intermediates between compartments. Diterpenes number more than 12,000 structures and whose syntheses are prime examples of membrane-spanning plant metabolic pathways. Plastid-localized diterpene synthases cyclize the universal precursor geranylgeranyl diphosphate (GGDP) into various diterpene olefins that are then trafficked into the endoplasmic reticulum (ER) for further oxidation reactions by ER-resident cytochrome P450s. Fifty-three plastid envelope transporters have been identified and characterized and all but two move polar metabolites and most notably, none have been identified for non-polar diterpene olefins. A previous study showed that plastid-localized tocopherol and carotenoid precursors were accessible in a bidirectional fashion from the ER lumen, suggesting that a novel interface exists between plastids and the ER that could allow access of non-polar compounds between the two organelles independent of transporters. In this dissertation, the accessibility of diterpene olefins between the plastid and ER was studied using two diterpene pathways, the gibberellin (GA) and diterpene resin acid (DRA) pathways. In Chapter 2, the accessibility of ent-kaurene synthesized in the ER lumen by outer envelope membrane-localized kaurene oxidase is tested by retargeting both plastid-localized copalyl diphosphate synthase (CPS) and kaurene synthase (KS) into the ER in Arabidopsis mutant backgrounds lacking both activities. Access to ER-synthesized ent-kaurene was evidenced by complementation of the dwarf mutant phenotypes. Surprisingly, the polar intermediate, ent-copalyl diphosphate (ent-CDP) was also shown to be accessible in a unidirectional fashion from the ER back into the plastid. This was observed by complementation of the dwarf phenotype of the CPS mutant, ga1-6, by ER-targeted CPS. Localization studies of ER-targeted CPS-YFP protein in transgenic lines showed that ER:CPS-YFP was correctly targeted to the ER lumen. Metabolite analysis of various GA intermediates showed levels in the ER:CPS-YFP lines similar to WT. In Chapter 3, the accessibility of conifer-specific diterpene olefins, abietadiene and isopimaradiene by ER-localized CYP720B4 was investigated. These diterpene olefins were produced in stable Arabidopsis transgenic lines by overexpressing plastid-localized abietadiene and isopimaradine synthases. Expression of these conifer-specific bifunctional diterpene synthases however, only resulted in extremely low levels of the corresponding diterpene olefins. The low levels of the bifunctional diterpene synthase proteins produced, coupled to the apparent inability of these proteins to form productive complexes with the existing GGDP-producing machinery in Arabidopsis are the most likely explanations for the low levels of diterpene olefins produced. As a whole, the work in this dissertation provided more insight into the transport of ent-CDP and ent-kaurene in the GA pathway and that the transport processes for the two compounds are most likely mediated by two separate mechanisms.
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