Transcription factor movement and tissue patterning in Arabidopsis root meristem

Abstract

Cell-cell communication is key to coordinated cellular functions in multicellular organisms. In addition to the signaling molecules found in animals, plants also frequently recruit mobile transcription factors to deliver positional information. The best studied example is SHORT-ROOT (SHR), a transcription factor which moves outward from the central vasculature to specify the cell fates of quiescent center (QC), the stem cell cortex/endodermis initial and its daughter (CEI/D) and endodermis in the adjacent cell layer in the root of the model organism Arabidopsis thaliana. SHR is required together with its downstream target SCARECROW (SCR) for the formative divisions of CEI/D and separation of endodermis and cortex. Intensive studies have revealed that SHR intercellular mobility is linked to its subcellular localization, while formative divisions at the CEI/D position is determined by a regulatory network involving SHR and SCR. Despite these advances, additional regulatory mechanisms are needed to fully explain the regulation of SHR movement and action range. SHR movement is regulated by a zinc finger protein JACKDAW (JKD). We described the function of three JKD homologs and members of the “BIRD protein family” – BALDIBIS (BIB), MAGPIE (MGP) and NUTCRACKER (NUC) – and observed that they constrain SHR movement through nuclear retention while fine-tuning transcription of key SHR targets including SCR and CYCLIN D6 (CYCD6). JKD and the studied BIRD proteins promote tissue specifications to continuously stabilize tissue boundaries in the root meristem, highlighting the developmental plasticity in plants. SHR, SCR and the BIRD proteins are largely present in the QC, CEI/D and endodermis, yet different cell types are specified by their interplay. They are also likely to form protein complexes thanks to their inter-binding capacity. We hypothesized that they might form distinct protein complexes in different cells to precisely regulate their behaviors. We optimized the imaging technique Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) for Arabidopsis roots, and observed that SHR-SCR interaction is enriched in the CEI/D. We then confirmed that enhanced SHR-SCR interaction correlates with formative divisions, while JKD binds SHR and SCR in spatially complementary manners to repress undesired divisions outside CEI/D. Binding competition assays suggest that SHR, SCR and BIRD proteins are incorporated in higher order protein complexes. Together, these data indicate that differential protein interactions are associated with different cell fate specifications, and suggest how multicellular organisms might employ a limited set of regulatory factors to trigger various developmental processes. JKD can act as a guiding molecule for SHR into specific subnuclear bodies. We generated two JKD mutant variants with opposite effect on SHR regulation: one enhances SHR subnuclear body localization while repressing SCR and CYCD6 expression, the other fails to enrich SHR into subnuclear structures but enhances target transcription. Our data provide evidence of JKD subnuclear compartmentalization as a means to regulate target gene expression. We also described the function of SCARECROW-LIKE23 (SCL23), the closest homolog of SCR in Arabidopsis. SCL23 is also a mobile protein and that it restricts SHR movement without nuclear retention, adding more complexity towards SHR regulation during root development.