Ubiquitin E3 Ligases and Cell Polarity in Arabidopsis

Higher plants are characterized by a common blueprint, with main growth axes oriented upwards and downwards. Establishment of these growth axes already occurs during very early stages of plant development and is perpetuated throughout the entire life cycle. Control of directional growth is under tight control, responding to developmental and environmental signals, allowing for flexible adjustments in overall development. Several decades ago, the plant growth regulator auxin has been identified as key regulator of polarity establishment. The small molecule is produced in dividing tissue, from where it is actively transported throughout the entire plant body, thereby influencing a multitude of developmental processes. Transport of the growth regulator across cellular plasma membrane boundaries involves activity of a plant-specific family of membrane proteins, mediating auxin release from the cells interior into its surroundings, from where it is taken up by adjacent cells. These PIN-FORMED (PIN) membrane proteins often times exhibit an asymmetric distribution, restricted to defined portions of the plasma membrane. Such polarity in PIN distribution has been found to influence directionality of the auxin flow through cell files. Importantly, PIN localization at membrane domains is not static but may vary throughout plant development, allowing for adjustments in directionality of auxin flow, thereby influencing adaptations in plant development. Understanding mechanisms of PIN protein sorting and distribution is matter of intense research and also subject of this proposal. My lab identified a small group of proteins in the model plant Arabidopsis thaliana that belong to the class of ubiquitin ligases, catalyzing the reversible attachment of the small protein ubiquitin onto target proteins. Target modification by ubiquitin has been shown to influence protein function, stability and intracellular distribution, with wide-ranging effects on cellular processes. In case of aforesaid Arabidopsis ubiquitin ligases, we detected a polarity shift in the localization of a PIN protein from the upper to the lower side of root cells, specifically in mutant plants, deficient in the function of these ubiquitin ligases. Alterations in PIN distribution coincide with severe defects in auxin distribution, also manifested in aberrant root growth, underlining the importance of correct PIN protein positioning for plant development. In this project, we will determine mechanisms by which ubiquitin ligases affect polar protein sorting, including the identification of targets proteins and their function in the regulation of PIN distribution. This, together with genetic and cell biological approaches suitable for integration of the ubiquitin ligases into established signaling and protein sorting networks, should identify so far unknown mechanisms of polarity control in higher plants.

Plants as the primary source of any living matter, are continuously exposed to environmental signals and stimuli, stimulating or suppressing plant development and hence vitality. To cope with these factors, plants have invented a plethora of mechanisms, allowing to adjust and optimize plant performance, even under very stressful conditions. Plant adaptive growth responses are frequently governed by hormonal signaling events, with auxin representing one of the most versatile plant hormones characterized to date. Key to the activity of auxin in controlled morphogenesis and adaptive growth responses is its tightly regulated, directional transport throughout the entire plant body. Directionality of auxin transport in turn is determined by the asymmetric, polar localization of plant-specific PIN proteins, which transport auxin from a cell's interior into the extracellular space, from where it is taken up by neighboring cell(s). Highly polarized cellular auxin efflux in entire cell files, thereby defines the direction and amplitude of hormonal transport throughout plant tissues and organs. As a consequence, adjustments in the distribution of PIN proteins will influence the direction of auxin transport and a multitude of developmental processes that are controlled by auxin. Our research characterized a so far unknown family of plant proteins that are intimately involved in the control of PIN distribution. Specifically, a loss of functionality of these so-called WAV3 proteins results in a cellular polarity shift in the distribution of selected PINs, which then no longer accumulate at the upper/apical side of cells - to promote auxin transport in the upward direction, but relocate to the lower/basal side of these cells instead. The WAV3-type proteins do show a polar localization themselves, accumulating at the basal cellular domain, a localization that seems to be crucial for the correct sorting of PIN proteins designated to accumulate at the apical cellular domain. In addition, we managed to identify some fundamental mechanisms by which WAV3 proteins impact on the sorting of PIN proteins, as a central outcome of this project. Furthermore, WAV3 distribution was found to exhibit some dynamics itself, which in turn appears to be crucial for the dynamic redistribution of PIN proteins in adaptive growth responses. Elucidating mechanisms by which such dynamics in WAV3 protein localization guides the distribution of PIN proteins is subject of another currently ongoing project funded by the FWF, and should result in essential insights into mechanisms, by which plants adjust their growth in response to environmental signals.