Stem cells have fascinated humans for a long time because of their fundamental importance in building and sustaining many forms of life. Stem cells are able to divide and maintain themselves while producing new cell types. Like animal stem cells, plant stem cells are a fascinating research object. They provide the cells for plant growth and allow many plants to continue growing throughout their lives, thereby producing enormous amounts of plant biomass on this planet. In this study, I specifically focussed on the plant stem cells that produce wood and bast. These stem cells form a cylindrical stem cell niche, the vascular cambium, which is found in the root, the stem and the tissue that connects the root and stem, the hypocotyl. In my analyses, I focused on the hypocotyl of the model plant Arabidopsis thaliana, since wood and bast production is very active there and the tissue patterning resembles the patterning of the vascular cambium of trees.
A vascular cambium stem cell produces both, wood progenitor cells inward and bast progenitor cells outward. Thus, it needs to “choose” between the following cell fates: becoming a wood progenitor cell, becoming a bast progenitor cell, or remaining a stem cell. The basis on which this first cell fate decision is made has not yet been clarified. To better understand this decision-making process, in a first step I mathematically modelled gene regulatory networks in collaboration with the Mironova laboratory. This has shown that a network of just three genes that regulate each other in a specific way is sufficient to bring about the three cell fate possibilities mentioned above. I then hypothesized that this network could consist of two mutually inhibitory auxin and cytokinin signalling pathway components. Furthermore, these components could be additionally regulated by WUSCHEL-RELATED HOMEOBOX4 (WOX4), an important transcription factor for stem cell regulation in the vascular cambium. Integrated into a 1D model, this network generated bidirectional growth. Using in planta analysis, I was able to show that both auxin and cytokinin signalling levels are low specifically in the vascular cambium. To test my hypothesis, I investigated which genes of the auxin and cytokinin pathways are regulated by WOX4, also in comparison to WUSCHEL, a stem cell regulator of the shoot apical meristem. In the course of my investigations, I identified specific type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), which negatively regulate cytokinin signalling, as putative WOX4 targets. I found that the expression of the type-A ARR genes ARR5, ARR6, ARR7 and ARR15 is reduced in the hypocotyl vascular cambium. Furthermore, I observed that the expression of ARR6 and ARR15 is downregulated by WOX4. WUSCHEL also exerts this type of regulation on type-A ARRs in the shoot apical meristem and therefore this could represent a general concept for the regulation of plant stem cells. In addition, findings in this study propose that WOX4 itself is regulated by cytokinin via the DNA BINDING WITH ONE FINGER 2.1 (DOF2.1) protein. Importantly, when ARR7 and ARR15 are mutated, the cell fate decisions of vascular cambium stem cells are altered and more wood cells are produced than in wild type plants. In conclusion, this study suggests that a mechanism of cell fate decision making in vascular cambium stem cells is based on the regulation of cytokinin signalling by WOX4 and by type-A ARRs
