Molecular basis of root growth inhibition by auxin

Plants differ from animals in many aspects, but one of the most striking differences is that plants do not move. Also the cells of plants are immobile and bound to their neighboring cells throughout their lifespan. The shapes and patterns of plants emerge from a precise control of cell division orientation and the amount and direction of cellular growth. Plant cells have strong cell walls and are highly pressurized (turgor pressure); the pressure in the cells can reach up to 10 bar which can be compared to inflated tires of racing bicycles. In order to grow, plant cells have to regulate the balance between this high pressure and the properties of the cell walls. The plant hormone auxin is a crucial player in this regulation. If we apply auxin to above-ground organs, the cells will soften the structure of their cell walls and start to grow. On the other hand, when applied to roots, auxin stops cell growth nearly immediately. When plants are turned out of their normal gravity position the hormone auxin accumulates on lower sides of both, shoots and roots. Therefore the opposite cellular response to auxin is responsible for the fact that above ground organs grow upwards, while the roots grow downwards. In the proposed project, we aim to uncover the molecular mechanisms of how roots inhibit their growth after auxin application. Even though this is one of the longstanding questions in plant research, it has not been answered completely so far. We will do so by establishing a microfluidic system where we can treat the roots of the model plant Arabidopsis with desired concentrations of auxin and observe the cellular response at the same time using a fluorescence microscope. We will also identify molecular actors of auxin-induced growth inhibition by analysis of the protein modifications that occur after auxin treatment. Furthermore, we will find out which genes execute the auxin response by using the next-generation RNA sequencing. Studying the molecular pathways by which plant cells regulate growth inhibition and growth stimulation will help us understand how plant cells perceive and tune the fine balance between the cell wall properties and the turgor pressure one of the essences of being a plant.

This project aimed at understanding the molecular basis of root gravitropism the ability of roots to follow the gravitational field in the search for water and nutrients. Roots perceive the gravitational stimulus in their very tip in the cells of the so-called columella and then transmit the information about the change of gravity direction using the phytohormone auxin to the cells of the epidermis. Auxin causes a rapid inhibition of elongation in the responding epidermal cells, and this way the root turns downwards. Despite this phenomenon being one of the most studied effect of auxin, the mechanism of the growth inhibition is not understood. In order to perform the study, we developed a methodological framework that consists of a dedicated confocal laser scanning microscope where the roots can grow in the natural vertical position equipped with automatic root tip tracking and a microfluidic platform that enabled us to analyse the reaction of roots to stimuli in a very high spatio-temporal resolution. We analysed the reaction of roots to application of auxin in real time and discovered that the speed and dynamics of the reaction rules out the current understanding of the action of this phytohormone: roots reacted extremely quickly to auxin addition, but at the same time started to grow immediately after removal of the hormone. This is not consistent with the current understanding of the auxin perception and signalling pathway, which supposedly triggers changes in the transcription of auxin-responsive genes. We confirmed this hypothesis by pharmacological treatments, mathematical modelling, genetics and synthetic biology approach. We therefore postulate a novel signalling branch of the auxin perception pathway which is operational during root gravitropism. These results have implications beyond the problematics of root gravitropism, because the auxin perception pathway is crucial for virtually all aspects of the life of plants.