A Genetic Study of Cadaverine Response Reveals Crosstalk between Cadaverine and Putrescine Pathways in Arabidopsis thaliana

摘要

Cadaverine derives from lysine in a pathway distinct from that of the well-characterized ornithine- or arginine-derived polyamines. Also known as 1,5-diaminopentane, cadaverine is positively charged at physiological pH (5.5 vacuole, 7.5 cytosol), and has ample opportunity to interact with negatively charged cellular components, such as proteins, membranes, and nucleic acids. Despite a multitude of studies in bacterial systems, cadaverine has garnered little attention in plant research. We have evidence that exogenous cadaverine causes disparate phenotypes in different species. We also know that the endogenous cadaverine level of different plant species varies widely. This thesis addresses the genetic pathways that regulate cadaverine response in Arabidopsis thaliana.;In chapter 1, we provide an overview of cadaverine metabolism and function in plants, with focus on the mechanisms that mediate cadaverine biosynthesis, conjugation and conversion into alkaloids, and its catabolism. We also describe the potential sources of environmental cadaverine for plants, and discuss the role of cadaverine in plant development and environmental stress response.;In chapter 2, we discuss the results of a genetic screen for cadaverine-response mutants, identifying a copper amine oxidase gene, CuAO3, as contributing to the regulation of root growth response to cadaverine. We show that two independent mutations in this gene, cdr7 and cuao3-1, confer increased resistance to cadaverine. Interestingly, these mutations also confer increased sensitivity to putrescine, an arginine-derived diamine in Arabidopsis. We show the cadaverine-resistance phenotype displayed by these cuao3 mutants to be associated with decreased cadaverine levels, whereas putrescine levels are enhanced. Considering the demonstrated role of CuAO3 in putrescine catabolism, the data suggest the existence of unexpected cross-regulation between the putrescine and cadaverine metabolic pathways, a model that seems consistent with the demonstration of altered root growth responses to distinct metabolites in the cadaverine and putrescine pathways.;To address the role of environmental cadaverine in Arabidopsis, chapter 3 reports on investigations of the roles played by two transporters, PUT2 and PUT5, in cadaverine response. T-DNA insertional mutants of PUT5 are shown to display increased root-growth resistance to cadaverine, whereas mutations in PUT2 result in hypersensitivity. Published data has previously localized PUT2 to both the Golgi (Li et al., 2013) and chloroplast (Ahmed et al., 2017). Transient expression experiments in tobacco using a PUT5-YFP transgene demonstrate protein association with the endoplasmic reticulum. Our data suggest a role for PUT5 in cadaverine targeting to functional compartments in plant cells, whereas PUT2 may contribute to cadaverine sequestration away from their site(s) of action. Surprisingly, put2-1 put5-2 double mutants displayed the cadaverine hypersensitive phenotype associated with put2-1, suggesting PUT2 might function earlier in the pathway.;Overall, our data provide interesting new information that expand our understanding of the complex interplay that exists between distinct polyamine metabolic pathways in plants, and also suggest that distinct transporters may contribute to polyamine response in different ways, either by favoring the formation of an active pool in plant cells, or by sequestering it away from their cellular site of action.;Two appendices in this thesis also address the development of interesting new tools for the study of polyamine function in plants. In Appendix I, we detail the development of an inducible system that allows spatially and/or temporally controlled increases in cadaverine biosynthesis in plant organs. In Appendix II, we report on the discovery of an important role played by light in modulating the penetrance of the root-growth response to cadaverine phenotype displayed by the cdr7 and cuao3-1 mutants. These systems will be useful in studies of the molecular mechanisms that govern cadaverine metabolism, transport and response in plants.