Both plants and animals can regulate patterning through developmental clocks that involve oscillating gene expression. In Arabidopsis thaliana, the Root Clock determines organ spacing along the primary root axis by establishing prebranch sites (PBS) through oscillating gene expression (Figure 1A) approximately every 6 hour. The mechanism driving the oscillations and the periodicity of the Root Clock is unknown. Oscillations in gene expression occur as propagating waves in the oscillation zone (OZ) in two opposite phases: in phase and in antiphase based on expression of the DR5::Luciferase auxin-response reporter. When expression of in-phase genes is activated in the OZ, the expression of antiphase genes is repressed and vice versa. Intriguingly, periodicity of the root clock can vary under specific environmental conditions or by supplementation with the phytohormone auxin.

In this work led by the CBGP research groups “Root organogenesis, regeneration and rooting”, directed by Dr. Miguel A. Moreno-Risueno, and “PlantDynamics lab”, directed by Dr. Krzysztof Wabnik, the core molecular mechanism of the Root Clock and how it is modified by exogenous cues such as auxin and gravity has been identified. The key elements of the oscillator (the transcription factor AUXIN RESPONSE FACTOR (ARF) 7, its auxin-sensitive inhibitor IAA18/POTENT and auxin) form a negative regulatory loop circuit in the OZ (Figure 1B). First, we identified the mutant potent in a mutagenesis screen. Gene expression oscillations in potent lack the typical 6-hour oscillatory behavior causing abnormal PBS spacing and branching (Figure 1C). potent is a dominant mutation in the DII domain of the Aux/IAA18 gene resulting in increased protein stability and abnormal auxin signaling. Aux/IAA proteins are inhibitors of ARFs. We found that the loss-of-function mutant in ARF7 showed (similarly to potent) absence of the characteristic oscillations resulting in more PBS formation (Figure 1D). Furthermore, IAA18/POTENT and ARF7 form heterodimers in the OZ, which de-represses the oscillations because IAA18/POTENT inhibits ARF7 activity in the OZ.

Through multilevel computer modeling, we explain how this circuit generates gene expression oscillations and how it coordinates with cell division and growth to create the periodic pattern of organ spacing. The model tracks the growth of xylem pole pericycle or pericycle in time and space as priming occurs in these tissues (Figure 1E). The model simulations showed a dynamic wave of DR5/in-phase genes originating at the basal meristem and moving shootwards through the OZ every 5-6 hours, demonstrating that the oscillatory waves of gene expression are an emergent property of this system. Biological clocks can be entrained by external or environmental cues, and our simulations demonstrated that external auxin stimuli can lead to entrainment of the Root Clock as it is observed during gravistimulation. In agreement with the model predictions gravistimulated wild-type roots displayed an increased frequency in PBS formation, whereas areas with fused PBS were observed in potent and arf7 mutants (Figura 1F), confirming a reduced capacity of these mutants to respond to external changes.

Our work demonstrates the molecular mechanism defining a biological clock and how a gene oscillator can be positioned in a growing organ to create a robust pattern of periodic organogenesis while maintaining the ability to respond to external stimuli.

Original Paper:

Perianez-Rodriguez, J., Rodriguez, M., Marconi, M., Bustillo-Avendaño, E., Wachsman, G., Sanchez-Corrionero, A., De Gernier, H., Cabrera, J., Perez-Garcia, P., Gude, I., Saez, A., Serrano-Ron, L., Beeckman, T., Benfey, P.N., Rodríguez-Patón, A., del Pozo, J.C., Wabnik, K., Moreno-Risueno, M.A. 2021. An auxin-regulable oscillatory circuit drives the root clock in Arabidopsis. Science Advances 7, eabd4722. DOI: 10.1126/sciadv.abd4722

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