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Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate–binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission.

Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C ₃ side chain by two carbon units, which can occur by a non–β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes.

• The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. • We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. • CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. • CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent.

In addition to proteins, L -phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network. Phenylalanine is synthesized in plant chloroplasts and is then exported to the cytosol, where it is a precursor for various secondary metabolites. Here, the authors identify PhpCAT as a plastid phenylalanine transporter required to maintain metabolic flux in petunia.

Sessile organisms, such as plants, have developed intricate means of responding and interacting with their environment in order to grow, reproduce, and survive environmental stresses such as attacks by other organisms and competition for resources with neighboring organisms. Plant volatile organic compounds (VOCs) play vital roles in resolving these evolutionary constraints associated with the sedentary nature of plants by attracting pollinators and seed dispersers necessary for reproduction. VOCs also mediate plant-plant interactions and provide defense against biotic stresses (pathogens, predators, and herbivores) and abiotic stresses (oxidative stress, high temperature stress, drought). Beyond the importance of VOCs to plants, humans have used VOCs for centuries as perfumes, therapeutics, food additives, and contribute to the flavor and aroma of fruits and vegetables. Chapter 1 of this dissertation offers a brief overview of plant VOCs, their functions, and biosynthetic pathways. The factors influencing their emission and the long-standing diffusion model of volatile release from plant cells are also discussed. Chapter 2 challenges the diffusion model and provides evidence for the involvement of active transport in the passage of VOCs across the plasma membrane. Downregulation of an ATP-binding cassette (ABC) transporter, PhABCG1, by RNA interference (RNAi) in Petunia hybrida flowers led to a decrease in volatile emission and accumulating of the internal pools of volatiles to toxic levels in the plasma membrane. In addition, PhABCG1 was shown to directly transport benzenoid volatiles using Nicotiana tabacum BY2 cells overexpressing PhABCG1. Together, these results alter the default assumption that VOCs simply diffuse out of cells. Chapter 3 completes the identification of the biosynthetic genes in the peroxisomal β-oxidative pathway of benzoic acid (BA) biosynthesis by the identification of a petunia cinnamoyl-CoA hydratase-dehydrogenase (PhCHD). Kinetic analysis of recombinant PhCHD shows that it converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA in vitro. Furthermore, downregulation of PhCHD in petunia flowers, using an RNAi approach, led to a decrease in benzoyl-CoA (BA-CoA), BA and other benzenoid derived volatiles further demonstrating the involvement of this gene to the peroxisomal β-oxidative pathway of BA biosynthesis. Lastly, Chapter 4 investigates the possible mechanisms of transport of the final product BA-CoA, of β-oxidative BA metabolism out of peroxisomes. Since the CoA moiety is membrane impermeable and BA-CoA thioesterase activity is enriched in purified peroxisomes, we hypothesized that BA-CoA is converted to BA by a thioesterase prior to transport or diffusion across the membrane and then reconverted to BA by a CoA ligase such as Ph4CL1 (petunia 4-coumarate: CoA ligase 1) or BZL1 (benzoate: CoA ligase) or by an unknown membrane associated ligase. Characterization of recombinant PhTE1 shows that it efficiently converts several hydroxycinnamoyl-CoA thioesters, including BA-CoA, to their free acids. Also, downregulation of PhTE1 led to an increase in the levels of BA-CoA and its derived volatiles, suggesting that BA-CoA is most likely transported out of peroxisomes. Furthermore, the levels of volatile phenylpropenes, anthocyanin and lignin were also altered suggesting cross-talk between the ?-oxidative and the general phenylpropanoid pathways. Together, these results suggest the auxiliary roles thioesterases play in β-oxidative metabolism.

Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C3 side chain by two carbon units, which can occur by a non-β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid [arrow right] cinnamoyl-CoA [arrow right] 3-hydroxy-3-phenylpropanoyl-CoA [arrow right] 3-oxo-3-phenylpropanoyl-CoA [arrow right] BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes. [PUBLICATION ABSTRACT]