Novel regulators of cellular auxin homeostasis

The phytohormone auxin is an essential regulator for plant growth and development. Local auxin maxima and minima are formed throughout plant development and induce multiple developmental and physiological processes. Decades of intensive research revealed the mutual importance of auxin metabolism and intercellular cell-to-cell transport for the regulation of spatiotemporal auxin distribution. Just recently putative intracellular auxin carriers, such as PIN-FORMED (PIN)5/PIN8 and PIN-LIKE (PILS)2/PILS5, were discovered and seem to limit nuclear auxin signalling via an auxin sequestration mechanism. PILS proteins define cellular sensitivity to the phytohormone auxin. Moreover, these putative auxin carriers at the endoplasmic reticulum might provide a link between auxin compartmentalization and auxin conjugation-based metabolism. Mutant analyses suggest that putative intracellular auxin transporters contribute to various developmental aspects, such as gametophyte development, postembryonic organogenesis and cellular growth regulations. Here we propose to use a combination of forward genetics and chemical genomics to reveal unbiased insight into PILS-dependent regulation of cellular auxin homeostasis. In order to team these approaches, we have used the same screening strategy (PILS suppressor and enhancer screen) in a forward genetic and a chemical screen. We aim to identify and characterize upstream and downstream regulators of PILS activity. We have identified mutants that suppress or enhance specific PILS-dependent phenotypes. In this study, we aim to identify and characterize some of the underlying gene products affecting PILS activity. This grant has the potential to reveal completely novel molecular insight into PILS activity and cellular auxin homeostasis regulation. This knowledge will broaden our understanding of auxin biology and how subcellular mechanisms could be used to guide plant development. Furthermore, we aim to characterize pharmacological tools to interfere with PILS-dependent processes. We identified compounds that either suppress or enhance PILS-dependent phenotypes. We will in depth characterize the isolated compounds and will use them as a tool to characterize molecular pathways affecting PILS function. Possible biotechnological application of the compounds will be evaluated. Compound classification, using genetic and transcriptomic tools, is much faster compared to genetic mutant characterization. The identified mutants and compounds will be initially characterized and mutant sensitivity to the compounds will be assayed to functionally group the identified mutants and compounds. This combinatory approach will allow us to assess the mutant and compound specificity at an early stage of the project. This project will very likely reveal novel and unanticipated insight into cellular mechanism of cellular auxin homeostasis at the endoplasmic reticulum.

Plant derived materials are utterly important for our everyday life. Nevertheless, we still have only a limited understanding of basic processes controlling plant growth and development. The phytohormone auxin is a central growth regulator and is virtually involved in every developmental aspect of a plant life cycle. The auxin receptor resides in the nucleus, where it regulates gene expression in an auxin-dependent manner. There are however mechanisms that can suppress the cellular response to auxin. Our research team has identified the novel family of putative auxin carriers, which we termed the PILS proteins. They localise to the endoplasmic reticulum (ER) and presumably transport auxin into its lumen to limit the diffusion of auxin into the nucleus. To further our knowledge on this growth relevant mechanism, we isolated mutants that modulate PILS-dependent architectural traits. In this project, we realised that brassinosteroid, another plant hormone, regulates PILS genes, which allows this hormone to modulate auxin responses. This hormonal crosstalk mechanism is important for organ growth rates in Arabidopsis thaliana. Another mutant pointed to the regulation of PILS protein turnover at the endoplasmic reticulum. We isolated a ubiquitin ligase, which resides in the peri-nuclear region, where it presumably marks PILS proteins for degradation, which presumably affects auxin diffusion rates into the nucleus. Yet another mutant may hint at a mechanism, revealing how auxin signalling controls the metabolic state of a cell. In this project, we have isolated various mutants that will provide a substantial mechanistic advance in plant growth regulation.