Abstract:
As the principal photosynthetic organ, a leaf commonly develops as a thin, flat lamina optimised for light capture and gas exchange. The flattened architecture, while seemingly simple, presents a developmentally challenging problem. How can a microscopic bud acquire and retain a planar shape as it grows into a macroscopic structure? While the significant components promoting adaxial and abaxial (ad-ab) identities are known, how these come together to first specify polarity and then to propagate this to sustain formation of a planar leaf, while allowing for developmental flexibility, remained unclear. Combining time lapse imaging, cell lineage tracing, and molecular analysis, we show that the adaxial and abaxial determinants AS2 and KAN1 respectively, prepatterns the meristem periphery. This prepattern converts the uniform auxin input that drives organogenesis into an ARF3-dependent binary readout that distinguishes ad-ab domains. Upon emergence from this prepattern, the initial polarity resolves into a gene regulatory network (GRN) involving Transcription Factors (TF) and the small RNAs, miR165/166 and tasiARF. The latter function as morphogens, providing necessary positional information. Through theoretical and computational modelling, we have found the GRN to follow the organizing principles of a Turing system that dynamically adapts to keep the ad-ab boundary robust to perturbations and provide the flexibility needed for generating diversity in leaf shapes. In the GRN, miR165/166 is part of a sensitive node that needs to be accommodated to ensure robustness. Via in depth molecular and genetic analysis we show that genetic redundancy at the family level ensures spatiotemporal precision of miR165/166 function and generates excess small RNA. These are mechanisms that plants employ to safeguard miR165/166. At the individual gene level, redundant cis-regulatory modules (CRMs) within the MIR166A promoter ensure precise expression of MIR166A. This translates into phenotypic robustness generating a flat leaf at the organ level. Further, at a trans level, a complex network of TFs, involving activators and repressors with tissue-specificity or broad expression, and with multiple binding sites distributed across the promoter, generate tissue specificity of MIR166A and quantitatively regulate its levels at the shoot apex. This highlights potential general phenomena in morphogen regulation. Taken together, this thesis provides new insights into the establishment and maintenance of ad-ab polarity accommodating the formation of a flat leaf, as well as contributing to a wide range of other leaf shapes. Further, using ad-ab patterning as a model, I identify molecular mechanisms that safeguard the expression of sensitive network components as well as establishing the tissue specificity of key morphogens, ensuring robustness in patterning.
